Retrocyclins: antiviral and antimicrobial peptides

ABSTRACT

Retrocyclin peptides are small antimicrobial agents with potent activity against bacteria and viruses. The peptides are nonhemolytic, and exhibit minimal in vitro cytotoxicity. A pharmaceutical composition comprising retrocyclin as an active agent is administered therapeutically to a patient suffering from a bacterial and/or viral infection, or to an individual facing exposure to a bacterial and/or viral infection, especially one caused by the HIV-1 retrovirus or other sexually-transmitted pathogens.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with government support under grant numberAI22839 awarded by the National Institutes of Health. The government hascertain rights in the invention.

BACKGROUND

Natural polycationic antimicrobial peptides have been found in manydifferent species of animals and insects and shown to have broadantimicrobial activity. In mammals, these antimicrobial peptides arerepresented by two families, the defensins and the cathelicidins. Nearlyall of these peptides have membrane affinity, and can permeate andpermeabilize bacterial membranes, resulting in injury, lysis, and/ordeath to the microbes. In particular, the human peptides known asdefensins are produced by mammalian and avian leukocytes (e.g.neutrophils, some macrophages) and epithelial cells.

Three defensin subfamilies exist in vertebrates: alpha-defensins,beta-defensins, and circular (theta) minidefensins. All derive from anancestral gene that existed before reptiles and birds diverged, containsix cysteines, and have largely beta-sheet structures that arestabilized by three intramolecular disulfide bonds. RTD-1, a thetaminidefensin, was recently detected in bone marrow from the rhesusmonkey, Macacca mulatta. It had 18 residues and was circular, havingbeen formed by the fusion of two truncated alpha-defensin precursors(“demidefensins”) each of which contributed 3 cysteines to the maturepeptide. The cellular machinery responsible for processing theseprecursors remains operational in human leukocytes.

Alpha-defensins are largely beta sheet peptides that contain 29-35 aminoacid residues, including 6 cysteines that form three intramoleculardisulfide bonds. Because of the nature of the cysteine pairings, themolecules are effectively macrocyclic. Four of these α-defensins, HNP1-4, occur primarily in human neutrophils. HD-5 & 6 are found in Panethcells, specialized cells of the small intestine's crypts. Humanα-defensin genes contain three exons and two introns and are clusteredon chromosome 8p23. They encode preprodefensins that contain ˜100residues which encode a signal peptide, polyanionic propiece and theC-terminal defensin domain. Mature defensins are processed by sequentialproteolysis.

Beta defensins are generally larger than α-defensins (35-40 residues)and may also be more ancient, since they occur in birds as well asmammals. Beta defensins are expressed in many different types ofepithelial cells, and in some glands. In some cases, expression isconstitutive; in others, it is inducible. Several β-defensin genes arelocated on 8 p23, adjacent to the α-defensin genes—consistent with theircommon evolutionary ancestry. The disulfide pairing motif of betadefensins differs from that of α-defensins, however α and β-defensinshave generally similar shapes.

The three-dimensional structure of many defensins comprises a complexlyfolded amphiphillic beta-sheet, with the polar face formed by itsarginines and by the N- and C-terminal residues playing an importantrole in defining microbicidal potency and the antimicrobial spectrum.The antimicrobial effects of defensins are derived from their ability topermeabilize cell membranes and interact with viral envelopes, therebyexposing contents of the microorganism to the environment or abrogatingviral infectivity. (See Gudmundsson et al. (1999) J Immunol Methods232(1-2):45-54.) Antimicrobial peptides are reviewed by Hancock andLehrer (1998) Trends in Biotechnology 16:82.

In general, the antiviral activities of antimicrobial peptides have notbeen extensively investigated. Although studies have reported thatantimicrobial peptides, such as human neutrophil-derived defensins(α-defensins), are directly virucidal against herpes simplex virus(HSV), and adenovirus strains, only a few reports deal with anti-HIV-1activity. T22 and T140, analogs of polyphemusins (peptides fromhorseshoe crabs), are active in inhibiting HIV-1; replication throughbinding to the chemokine receptor CXCR4. However, these peptides onlyinhibit the T cell-tropic (T-tropic; X4) strains that utilize CXCR4 as acoreceptor for entry and they are ineffective against strains thatutilize CCR5 for entry (macrophage (M)-tropic “R5” viruses). Sincesexual transmission is largely attributed to R5 infection, the potentialof T22 and T140 as topical vaginal or rectal microbicides is limited.

One study indicated that protegrins (porcine-derived peptides) caninactivate HIV-1 virions. Another study showed that indolicidin, a 13amino acid peptide isolated from bovine neutrophils, was reproduciblyvirucidal against HIV-1 only at very high concentrations (333 μg/ml) ofpeptide. While the anti-HIV-1 activity of human α-defensins has not beenreported, certain structural and functional similarities exist betweenthe loop motifs of α-defensins and peptides derived from HIV-1 gp41 thatmay be required for viral fusion and infectivity.

Vaginal and rectal subepithelial stromal tissues are densely populatedwith dendritic cells (DC), macrophages and T-cells that express both CD4and the HIV-1 coreceptors, CXCR4 and CCR5. Mechanisms whereby HIV-1journeys across the mucosal epithelia are not clear, but may directlyinvolve the epithelial cells. Once the virus reaches the lamina propria,it can either directly infect macrophages or T-cells or adhere to orinfect DC whose traffic to the regional lymph nodes conveys them intosites of vigorous viral replication. A recent report suggests thatbinding of HIV-1 to DC is mediated by the C-type lectin DC-SIGN,independent of CD4 or chemokine receptors. Thus, mucosal factors whichmodulate steps in this process could affect the probability oftransmission of HIV-1 infection.

There is a clinical need for novel antiviral and antimicrobial agentsthat have low toxicity against mammalian cells. The present inventionaddresses this need.

Relevant Literature

Defensins are reviewed by Lehrer et al. (1992) Ann. Rev. Immunol.11:105-128. Other endogenous antimicrobials are reviewed in Schonwetteret al. (1995) Science 267:1645-1648; Schroder (1999) Cell Mol Life Sci.56:32-46 (1999); and Harwig et al. (1994) FEBS Lett 342:281-285.

Specific defensins are described in Tang et al. (1999) Science286:498-502; Zimmermann et al. (1995) Biochemistry 34:13663-13671; Liuet al. (1997) Genomics 43:316-320; and Palfree & Shen (1994) GenBankU10267; Polley et al. GenBank AF238378 disclose the sequence of Homosapiens chromosome 8p23 clone SCb-561 b 17.

Retrovirus infection and antiretroviral therapy are discussed in Wilsonet al. (1995) J. Infect. Dis. 172:88-96; Wong et al. Science278:1291-1295; and Yang et al.(1999) J. Virol. 73, 4582-4589.

SUMMARY OF THE INVENTION

Methods and compositions are provided for the use of retrocyclinpeptides. Retrocyclin peptides are small antimicrobial agents withpotent activity against viruses, e.g. enveloped viruses such asretroviruses; and bacteria. These circular peptides are nonhemolytic andgenerally exhibit little or no in vitro cytotoxicity. Retrocyclins areequally effective against growing and stationary phase bacteria, andthey retain activity against some bacteria in physiological, and highsalt concentrations. Studies indicate that retrocyclins are also capableof conferring immunity to human CD4⁺ cells against infection by HIV-1 invitro. In addition, other circular mini-defensins also find use asanti-viral agents, particularly against human retroviruses.

A pharmaceutical composition comprising retrocyclin or other circularmini-defensins as an active agent is administered to a patient sufferingfrom a viral infection. Alternatively, a pharmaceutical compositioncomprising retrocyclin or other circular mini-defensins or isadministered as a protective agent to a normal individual facingpotential exposure to HIV viruses or pathogenic microbes. Retrocyclin isalso effective at killing a variety of microbial organisms, in vivo andin vitro. Retrocyclin may be administered alone, or in combination withother bacteriocidal agents, e.g. antibiotics and/or other antiviralagents, and antiviral agents as a cocktail of effective peptides, etc.Retrocyclin-mediated killing is also useful for modeling and screeningnovel antibiotics.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Circular minidefensins reduce HIV-1 infection of H9 cells. HIV-Istrain IIIB (MOI=10⁻²) was incubated with 2.5×10⁵ H9 cells in thepresence or absence of 20 μg/ml RTD-1, RTD-2 or RTD-3. p24 antigenrelease was monitored by ELISA on days 3, 6, and 9. Assay sensitivity=10pg/ml.

FIG. 2. Sequence comparison of human and rhesus demidefensins. Thetranslated sequences of rhesus demidefensin-1 mRNA and human retrocyclinmRNA are shown. Solid circles () indicate a stop codon in thecorresponding cDNA. Vertical bars connect identical residues, and +signs connect similar residues. Residues represented in matureretrocyclin and RTD-molecules are boxed. The demidefensin-1 sequence(GenBank, AF184156) was derived from the monkey mRNA (not shown).

FIG. 3. Structural characterization of retrocyclin. (A) CD spectrumdemonstrating the similarity in structure between retrocyclin and RTD1,both at 0.5 mg/ml in a 1:1 mixture of trifluoroethanol in phosphatebuffered saline at pH 7.4. (B) shows a hypothetical model of retrocyclinmade by templating its sequence on the backbone of a similar peptidefrom porcine neutrophils, Protegrin-1 (PDB accession code: 1PG1). (D) isa cartoon version of (B), wherein arginines are black, cysteines aregrey and the other residues are identified by single letter code. (C) isa similar cartoon of rhesus RTD-1, indicating the similarity instructure with retrocyclin.

FIG. 4. Effect of salt on antibacterial activity of circularminidefensins. Human retrocyclin and monkey RTD-1 were tested againstour standard lab stains: E. coli ML-35p, P. aeruginosa MR 3007, L.monocytogenes EGD, and S. aureus 930918. The bars show MIC values±SEMvalues that resulted from 3-6 radial diffusion assays per organism andassay condition.

FIG. 5. Anti-HIV-1 activity of retrocyclin. Two strains of HIV-1 and twotypes of human target cells were used. The IIIB strain is T-cell tropic(X4) and utilizes the CXCR4 co-receptor for entry; the JR-CSF strain isM-tropic (R5) and uses CCR5 for entry. PBMC signifies CD4⁺-selectedperipheral blood mononuclear cells. Results indicate p24 antigenconcentration in pg/ml, as determined by quantitative ELISA assay at Day9 timepoint. (A) Two concentrations of retrocyclin (2 μg/ml, 20 μg/ml),20 μg/ml of the Rhesus circular defensin “RTD-1”, and 20 μg/ml of ahorseshoe crab-derived peptide “T140”, reported to only prevent X4infections, were tested in antiviral assays of against strain IIIB in H9cells (n=2-6 per peptide; error bars indicate SEM). (B) To confirm ourresults with primary human cells, similar assays were performedutilizing IIIB virus and CD4⁺PBMC or (C) JR-CSF virus and CD4⁺PBMC.Peptides were not cytotoxic at indicated concentrations, measured bytrypan blue exclusion. Average of duplicate experiments are reported forstudies with PBMCs. Assay sensitivity=10 pg/ml.

FIG. 6. Retrocyclin can inhibit HIV-1 spread when administered up to 24hrs post-infection. Primary CD4⁺PBMC were incubated with HIV-IIIB for 3hours in the absence (“control”, “t0”, “t3”, and “t24”) or presence (“t0only”) of 20 μg/ml retrocyclin. Cells were transferred to fresh R10-50media that was either supplemented immediately with 20 μg/ml retrocyclin(“t0”), or 3 or 24 hrs after transfer (“t3”and “t24”, respectively).“Control” and “tΦ only” were not supplemented after transfer. p24antigen was measured by ELISA as previously described.

FIG. 7. Mature retrocyclin, but not premature forms, inhibit HIV-1replication. H9 cells were incubated with HIV-IIIB (MOI=10⁻²) for 3hours in the absence or presence of 20 μg/ml retrocyclin in threeflavors: linear and reduced; linear and oxidized disulfide bonds; andthe mature form (cyclic and oxidized). Assay sensitivity is 10 pg/ml.

FIG. 8. Cytotoxicity of antimicrobial peptides against H9 cells.Retrocyclin, RTD-1 and PG-1 (a porcine-derived peptide with anti-HIV-1activity) were tested for cytotoxicity using an MTT assay for cellproliferation. Note that the EC₅₀ of Retrocyclin and RTD-1 were >100μg/ml, concentrations well above their antiviral concentration.

FIGS. 9A and 9B are graphs depicting the activity of retrocyclincongeners against HIV-1 strains.

FIGS. 10A and 10B are schematics depicting the structure of retrocyclin.

FIGS. 11A-C compares the antiretroviral activity of retrocyclin andRC-101 (20 μg/ml), by showing the p24 titers from day 9 CD4⁺PBMC(peripheral blood mononuclear cells) infected with HIV-1 strains IIIB orJR-CSF at the indicated MOI. RC-101 and retrocyclin were similarlyeffective in inhibiting HIV-1 replication at low MOI (A) and higher MOI(B).

FIG. 12. Adding retrocyclin directly with HIV-1-IIIB does not reduceinfection of H9 cells. Retrocyclin (2-200 μg/ml) was incubated withHIV-IIIB (MOI=10⁻²) diluted in R10 media prior to infecting H9 cells.p24 antigen release was measured by ELISA. Limit of detection=10 pg/ml.

FIG. 13. Retrocyclin and RC-101 inhibited the formation of HIV proviralDNA. Retrocyclin and RC-101 inhibited the formation of DNA from bothearly events (total HIV DNA) and later events (full-length HIV DNA) ofreverse transcription. Data are an average of 2 experiments, except forRC-101 (1 experiment). “HI virus” is a heat-inactivated virus controlfor background levels of viral DNA.

FIG. 14. Inactivation of HSV-1 and HSV-2 by retrocyclin and RC-101.Retrocyclin (left panel) and RC-101 (right panel) at the indicatedconcentrations were incubated with herpes simplex virus, type 1 (HSV-1)or HSV-2 for 2 hrs and then added to ME-180 cell monolayers. Cells wereincubated at 37° C. for 72 hrs, and cytotoxicity was measured with anMTT kit.

FIG. 15 depicts sequences of human, ape and monkey retrocyclins.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

Novel compositions and methods are provided for the use of retrocyclinsand retrocyclin analogs as therapeutic and/or prophylactic agents. Thepeptides are effective at killing a variety of microbial organisms bydirect microbicidal activity, and protect against viral infection by avirus by preventing viral uptake and/or blocking an early step in viralreplication. Retrocyclin(s) are administered alone or in combinationwith other active agents to a patient suffering from an infection in adose and for a period of time sufficient to reduce the patientpopulation of pathogenic microbes or viruses. Alternatively, apharmaceutical composition comprising retrocyclin or other circularmini-defensins or is administered as a protective agent to a normalindividual facing potential exposure to HIV viruses or pathogenicmicrobes. In addition, other circular mini-defensins, including RTD-1,RTD-2 and RTD-3 and variants of retrocyclin find use as anti-viralagents.

Specific treatments of interest include, without limitation: usingretrocyclin (e.g., RC-101) or a retrocyclin analog to prevent or treatinfection, for example by an enveloped virus, including envelopedretroviruses, more specifically by HIV-1, HIV-2 and related retrovirusesthat cause Acquired Immunodeficiency Syndrome (AIDS); aerosoladministration to the lungs of patients with cystic fibrosis to combatinfection or forestall the emergence of resistance to other inhaledantibiotics; instillation into the urinary bladder of patients withindwelling catheters to prevent infection; application to the skin ofpatients with serious burns; opthalmic instillation, directly or inophthalmic solutions, to treat or prevent infection; intravaginalapplication to treat bacterial vaginosis and/or prevent sexuallytransmitted disease such as HIV infection. The retrocyclins also mayfind use in the treatment of plant-pathogenic pseudomonads, inagricultural applications designed to prevent disease in and spoilage offood crops. The retrocyclins may be administered alone or in conjunctionwith other antiviral therapy.

The peptide form of retrocyclins provides a basis for furthertherapeutic development, by modification of the polypeptide structure toyield modified forms having altered biological and chemical properties.The native or modified forms are formulated in a physiologicallyacceptable carrier for therapeutic uses, or are otherwise used as anantimicrobial agent.

Retrocyclin Compositions

For use in the subject methods, a naturally occurring or syntheticretrocyclin may be used. As used herein, retrocyclins are cyclicpolypeptides comprising the amino acid sequence: X₁ X₂ X₃ X₄ X₅ X₆ X₇ X₈X₉ X₁₀ X₁₁ X₁₂ X₁₃ X₁₄ X₁₅ X₁₆ X₁₇ X₁₈

wherein X1 and X18 are linked through a peptide bond,

disulfide crosslinks are formed between at least one of: X₃ and X₁₆; X₅and X₁₄, and X₇ and X₁₂, usually between at least two of such pairs, andpreferably between the three pairs of amino acids, with the proviso thatwhen such a crosslink is present, the crosslinked amino acids are bothcysteines;

at least about three of amino acids X₁ to X₁₈ are arginine or lysine,and the number of arginine or lysine residues may be four or more, fiveor more, or six or more. Preferred residues for arginine or lysine areX₄, X₉, X₈, X₁₃, and X₁₈;

X₂, X₅, X₁₁, X₁₅ are preferably aliphatic amino acids, e.g. isoleucine,leucine, valine, phenylalanine, and alanine;

X₁, X₈, X₁₀ and X₁₇ are preferably glycine or alanine, usually glycine.

Retrocyclins are octadecapeptides that contain two linked nonapeptidesthat may be identical or different. A consensus nonapeptide has thesequence shown below, where the bolded and underlined residues areinvariant among the primate sequences identified herein. Substitutionsfound in the nonapeptide regions of other circular minidefensinprecursors are shown below the consensus nonapeptide.

Residue No 1 3 5 7 9 (SEQ ID NO:1) Consensus nonapeptide RC I C GRGI Cresidues 4-12) Variant   L RLRV Variant     T  F Variant     V Variant      R

From the consensus peptide and these variants, one can generate uniquenonapeptide sequences (herein termed n1, n2 . . . etc.). Thus, n1 couldbe linked to itself or any of the other nonapeptides (n1:n1, n1:n2,n1:n3 . . . etc.), to generate unique octadecapeptides. To continue theprocess, n2 could be linked to itself or to any other nonapeptide exceptn1, to generate additional unique octadecapeptides, and so forth.

Two naturally occurring human nonapeptide sequences are RCICGRGIC (SEQID NO:1, residues 4-12); and) RCICGRRIC (SEQ ID NO:74). The set ofnonapeptides derived from these sequences and variants (which are alsoprovided in the sequence listing as SEQ ID NO:19-64; and 9474-119) is asfollows:

Modi- Nona fica- SEQ ID NO Peptide # 1 2 3 4 5 6 7 8 9 tion* SEQ ID NO:19 1 R C I C G R G I C — SEQ ID NO: 20 2 R C L C G R G I C L3 SEQ ID NO:21 3 R C I C R R G I C R5 SEQ ID NO: 22 4 R C I C T R G I C T5 SEQ IDNO: 23 5 R C I C V R G I C V5 SEQ ID NO: 24 6 R C I C G L G I C L6 SEQID NO: 25 7 R C I C G R G V C V8 SEQ ID NO: 26 8 R C I C G R G F C F8SEQ ID NO: 27 9 R C L C R R G V C L3, R5 SEQ ID NO: 28 10 R C L C T R GI C L3, T5 SEQ ID NO: 29 11 R C L C V R G I C L3, V5 SEQ ID NO: 30 12 RC L C G L G V C L3, L6 SEQ ID NO: 31 13 R C L C G R G V C L3, V8 SEQ IDNO: 32 14 R C L C G R G F C L3, F8 SEQ ID NO: 33 15 R C I C R R G V CR5, V8 SEQ ID NO: 34 16 R C I C R R G F C R5, F8 SEQ ID NO: 35 17 R C IC T R G V C T5, V8 SEQ ID NO: 36 18 R C I C T R G F C T5, F8 SEQ ID NO:37 19 R C I C T L G I C T5, L6 SEQ ID NO: 38 20 R C I C V L G F C V5, L6SEQ ID NO: 39 21 R C I C R L G I C R5, L6 SEQ ID NO: 40 22 R C I C V R GV C V5, V8 SEQ ID NO: 41 23 R C I C G R G F C V5, F8 SEQ ID NO: 42 24 RC I C G L G F C L6, F8 SEQ ID NO: 43 25 R C I C G L G V C L6, F8 SEQ IDNO: 44 26 R C L C R L G I C L3, R5, L6 SEQ ID NO: 45 27 R C L C R R G VC L3, R5, V8 SEQ ID NO: 46 28 R C L C R R G F C L3, R5, F8 SEQ ID NO: 4729 R C L C T L G I C L3, T5, L6 SEQ ID NO: 48 30 R C L C G R G V C L3,T5, V8 SEQ ID NO: 49 31 R C L C T R G F C L3, T5, F8 SEQ ID NO: 50 32 RC L C V L G I C L3, V5, L6 SEQ ID NO: 51 33 R C L C V R G V C L3, V5, V8SEQ ID NO: 52 34 R C I C G R G I C L3, V5, F8 SEQ ID NO: 53 35 R C I C RL G V C R5, L6, V8 SEQ ID NO: 54 36 R C I C R L G F C R5, L6, F8 SEQ IDNO: 55 37 R C I C T L G V C T5, L6, V8 SEQ ID NO: 56 38 R C I C T L G FC T5, L6, F8 SEQ ID NO: 57 39 R C I C V L G V C V5, L6, V8 SEQ ID NO: 5840 R C I C V L G F C V5, L6, F8 SEQ ID NO: 59 41 R C L C G L G V C L3,R5, L6, V8 SEQ ID NO: 60 42 R C L C G L G I C L3, R5, L6, F8 SEQ ID NO:61 43 R C L C T L G V C L3, T5, L6, V8 SEQ ID NO: 62 44 R C L C T L G IC L3, T5, L6, F8 SEQ ID NO: 63 45 R C L C V L G V C L3, V5, L6, V8 SEQID NO: 64 46 R C L C V L G I C L3, V5, L6, F8 SEQ ID NO: 74 47 R C I C GR R I C — SEQ ID NO: 75 48 R C L C G R R I C L3 SEQ ID NO: 76 49 R C I CR R R I C R5 SEQ ID NO: 77 50 R C I C T R R I C T5 SEQ ID NO: 78 51 R CI C V R R I C V5 SEQ ID NO: 79 52 R C I C G L R I C L6 SEQ ID NO: 80 53R C I C G R R V C V8 SEQ ID NO: 81 54 R C I C G R R F C F8 SEQ ID NO: 8255 R C L C R R R V C L3, R5 SEQ ID NO: 83 56 R C L C T R R I C L3, T5SEQ ID NO: 84 57 R C L C V R R I C L3, V5 SEQ ID NO: 85 58 R C L C G L RV C L3, L6 SEQ ID NO: 86 59 R C L C G R R V C L3, V8 SEQ ID NO: 87 60 RC L C G R R F C L3, F8 SEQ ID NO: 88 61 R C I C R R R V C R5, V8 SEQ IDNO: 89 62 R C I C R R R F C R5, F8 SEQ ID NO: 90 63 R C I C T R R V CT5, V8 SEQ ID NO: 91 64 R C I C T R R F C T5, V8 SEQ ID NO: 92 65 R C IC T L R I C T5, L6 SEQ ID NO: 93 66 R C I C V L R F C V5, L6 SEQ ID NO:94 67 R C I C R L R I C R5, L6 SEQ ID NO: 95 68 R C I C V R R V C V5, V8SEQ ID NO: 96 69 R C I C R R R F C V5, F8 SEQ ID NO: 97 70 R C I C G L RF C L6, F8 SEQ ID NO: 98 71 R C I C G L R V C L6, V8 SEQ ID NO: 99 72 RC L C R L R I C L3, R5, L6 SEQ ID NO: 100 73 R C L C R R R V C L3, R5,V8 SEQ ID NO: 101 74 R C L C R R R F C L3, R5, F8 SEQ ID NO: 102 75 R CL C T L R I C L3, T5, L6 SEQ ID NO: 103 76 R C L C G R R V C L3, T5, V8SEQ ID NO: 104 77 R C L C T R R F C L3, T5, F8 SEQ ID NO: 105 78 R C L CV L R I C L3, V5, L6 SEQ ID NO: 106 79 R C L C V R R V C L3, V5, V8 SEQID NO: 107 80 R C I C G R R I C L3, V5, F8 SEQ ID NO: 108 81 R C I C R LR V C R5, L6, V8 SEQ ID NO: 109 82 R C I C R L R F C R5, L6, F8 SEQ IDNO: 110 83 R C I C T L R V C T5, L6, V8 SEQ ID NO: 111 84 R C I C T L RF C T5, L6, F8 SEQ ID NO: 112 85 R C I C V L R V C V5, L6, V8 SEQ ID NO:113 86 R C I C V L R F C V5, L6, F8 SEQ ID NO: 114 87 R C L C G L R V CL3, R5, L6, V8 SEQ ID NO: 115 88 R C L C G L R I C L3, R5, L6, V8 SEQ IDNO: 116 89 R C L C T L R V C L3, T5, L6, V8 SEQ ID NO: 117 90 R C L C TL R I C L3, T5, L6, F8 SEQ ID NO: 118 91 R C L C V L R V C L3, V5, L6,V8 SEQ ID NO: 119 92 R C L C V L R I C L3, V5, L6, F8 *residuemodifications are shown in this column. Nonapeptide # is a referencenumber for the nonamers.

Retrocyclins of interest include cyclic peptides derived from thepeptide sequence set forth in SEQ ID NO. 12, in particular a circularhomodimer comprising a dimer of the amino acid sequence SEQ ID NO: 12,aa 48-56. This retrocyclin has the structure (SEQ ID NO: 1):

G   I   C   R   C   I   C   G   R   G   I   C   R   C   I   C   G   R X₁ X₂  X₃   X₄  X₅  X₆   X₇  X₈  X₉   X₁₀ X₁₁ X₁₂ X₁₃  X₁₄ X₁₅  X₁₆ X₁₇X₁₈

Wherein X₁ and X₁₈ are joined by a peptide bond, X₂ and X₁₁; X₄ and X₉,and X₁₃ and X₁₈ are disulfide bonded.

Another retrocyclin of interest is the synthetic analog (SEQ ID NO: 2)

G   I   C   R   C   I   C   G   K G   I   C   R   C   I   C   G   R X₁ X₂  X₃  X₄   X₅  X₆  X₇   X₈  X₉  X₁₀  X₁₁ X₁₂ X₁₃  X₁₄ X₁₅ X₁₆ X₁₇ X₁₈

wherein X₁ and X₁₈ are joined by a peptide bond, X₂ and X₁₁; X₄ and X₉,and X₁₃ and X₁₈ are disulfide bonded. Other synthetic analogs, orcongeners, of retrocyclin are set forth in SEQ ID NO: 3-SEQ ID NO: 10.

The sequence of the retrocyclin polypeptides may be altered in variousways known in the art to generate targeted changes in sequence. Thepolypeptide will usually be substantially similar to the sequencesprovided herein, i.e. will differ by one amino acid, and may differ bytwo amino acids. The sequence changes may be substitutions, insertionsor deletions.

The protein may be joined to a wide variety of other oligopeptides orproteins for a variety purposes. By providing for expression of thesubject peptides, various post-translational modifications may beachieved. For example, by employing the appropriate coding sequences,one may provide famesylation or prenylation. In this situation, thepeptide will be bound to a lipid group at a terminus, so as to be ableto be bound to a lipid membrane, such as a liposome.

Modifications of interest that do not alter primary sequence includechemical derivatization of polypeptides, e.g., acetylation, orcarboxylation. Also included are modifications of glycosylation, e.g.those made by modifying the glycosylation patterns of a polypeptideduring its synthesis and processing or in further processing steps; e.g.by exposing the polypeptide to enzymes which affect glycosylation, suchas mammalian glycosylating or deglycosylating enzymes. Also embraced aresequences that have phosphorylated amino acid residues, e.g.phosphotyrosine, phosphoserine, or phosphothreonine.

Also included in the subject invention are polypeptides that have beenmodified using ordinary molecular biological techniques and syntheticchemistry so as to improve their resistance to proteolytic degradationor to optimize solubility properties or to render them more suitable asa therapeutic agent. Analogs of such polypeptides include thosecontaining residues other than naturally occurring L-amino acids, e.g.D-amino acids or non-naturally occurring synthetic amino acids.

The subject peptides may be prepared by in vitro synthesis, usingconventional methods as known in the art. Various commercial syntheticapparatuses are available, for example, automated synthesizers byApplied Biosystems, Inc., Foster City, Calif., Beckman, etc. By usingsynthesizers, naturally occurring amino acids may be substituted withunnatural amino acids. The particular sequence and the manner ofpreparation will be determined by convenience, economics, purityrequired, and the like.

If desired, various groups may be introduced into the peptide duringsynthesis or during expression, which allow for linking to othermolecules or to a surface. Thus cysteines can be used to makethioethers, histidines for linking to a metal ion complex, carboxylgroups for forming amides or esters, amino groups for forming amides,and the like.

The polypeptides may also be isolated and purified in accordance withconventional methods of recombinant synthesis. A lysate may be preparedof the expression host and the lysate purified using HPLC, exclusionchromatography, gel electrophoresis, affinity chromatography, or otherpurification technique. For the most part, the compositions which areused will comprise at least 20% by weight of the desired product, moreusually at least about 75% by weight, preferably at least about 95% byweight, and for therapeutic purposes, usually at least about 99.5% byweight, in relation to contaminants related to the method of preparationof the product and its purification. Usually, the percentages will bebased upon total protein. Genetic sequences encoding demi-defensins areprovided herein, e.g. SEQ ID NO: 4, 7 and 9.

In one embodiment of the invention, the antimicrobial peptide consistsessentially of a polypeptide sequence set forth in any one of SEQ ID NO:1-SEQ ID NO: 10. By “consisting essentially of” in the context of apolypeptide described herein, it is meant that the polypeptide iscomposed of the sequence set forth in the seqlist, which sequence may beflanked by one or more amino acid or other residues that do notmaterially affect the basic characteristic(s) of the polypeptide.

For some purposes of the invention, for example in the treatment and/orprevention of HIV infection, the active agent may be any one of thecircular minidefensins, e.g. human retrocyclins, RTD-1, RTD-2 and RTD-3.Cyclic minidefensins resemble protegrins, antimicrobial, sheet peptides.RTD-1 is derived from Macacca mulatta, and is a heterodimer containingtandem nonapeptide elements derived from the mature peptides set forthin SEQ ID NO: 15 and SEQ ID NO: 17. RTD-2 is a homodimer containing, intandem, two identical nonapeptide elements derived from the maturepeptide set forth in SEQ ID NO: 17. RTD-3 is a homodimer containing, intandem, two identical nonapeptide elements derived from the maturepeptide set forth in SEQ ID NO: 15.

All three RTD's are circular molecules with 18 residues and threeintramolecular disulfide bonds. Each RTD is formed by in vivo processingthat trims and splices two precursor peptides (“demidefensins”), each ofwhich contributes nine residues (including 3 cysteines) to the maturecyclic peptide. The 18RTD-1 residues derive from two differentdemidefensin precursors, RTD-2 and -3 have tandem 9 residue repeatsderived from a single demidefensin precursor.

Retrocyclin Coding Sequences

The invention includes nucleic acids having a sequence set forth in SEQID NO: 11, 120 or 122; nucleic acids that hybridize under stringentconditions, particularly conditions of high stringency, to the sequenceset forth in SEQ ID NO: SEQ ID NO: 11, 120 or 122; genes correspondingto the provided nucleic acids; sequences encoding retrocyclins; andfragments and derivatives thereof. Other nucleic acid compositionscontemplated by and within the scope of the present invention will bereadily apparent to one of ordinary skill in the art when provided withthe disclosure here. Genetic sequences of particular interest includeprimate sequences, e.g. human, chimpanzee, bonobo, orangutan, gorilla,etc.

Retrocyclin coding sequences can be generated by methods known in theart, e.g. by in vitro synthesis, recombinant methods, etc. to provide acoding sequence to corresponds to a linear retrocyclin polypeptide thatcould serve as an intermediate in the production of the cyclicretrocyclin molecule. Using the known genetic code, one can produce asuitable coding sequence. For example, the circular polypeptide ofretrocyclin (SEQ ID NO: 1) is encoded by the sequence (SEQ ID NO: 18)AGG TGC ATT TGC GGA AGA GGA ATT TGC AGG TGC ATT TGC GGA AGA GGA ATT TGC,but since the peptide is circular, it is somewhat arbitrary which codonis selected to be first, allowing this to be based on other criteria,e.g. relative efficiency in purification or cyclization of the predictedproduct. The polypeptide set forth in SEQ ID NO: 2 is encoded by asimilar sequence, wherein one of the arginine codons (AGA) issubstituted with a lysine codon (AAA or AAG).

The nucleic acids of the invention include nucleic acids having sequencesimilarity or sequence identity to SEQ ID NO: SEQ ID NO: 11, 18, 120 or122. Nucleic acids having sequence similarity are detected byhybridization under low stringency conditions, for example, at 50° C.and 10×SSC (0.9 M saline/0.09 M sodium citrate) and remain bound whensubjected to washing at 55° C. in 1×SSC. Sequence identity can bedetermined by hybridization under stringent conditions, for example, at50° C. or higher and 0.1×SSC (9 mM saline/0.9 mM sodium citrate).Hybridization methods and conditions are well known in the art, see,e.g., U.S. Pat. No. 5,707,829. Nucleic acids that are substantiallyidentical to the provided nucleic acid sequence, e.g. allelic variants,genetically altered versions of the gene, etc., bind to SEQ ID NO: SEQID NO: 11, 18, 120 or 122 under stringent hybridization conditions. Byusing probes, particularly labeled probes of DNA sequences, one canisolate homologous or related genes. The source of homologous genes canbe any species, e.g. primate species, particularly human; rodents, suchas rats and mice; canines, felines, bovines, ovines, equines, fish,yeast, nematodes, etc.

In one embodiment, hybridization is performed using at least 18contiguous nucleotides (nt) of SEQ ID NO: 1 and SEQ ID NO: 18, or a DNAencoding the polypeptide of SEQ ID NO: 1-10, 19-64, or 74-119. Such aprobe will preferentially hybridize with a nucleic acid comprising thecomplementary sequence, allowing the identification and retrieval of thenucleic acids that uniquely hybridize to the selected probe. Probes ofmore than 18 nt can be used, e.g., probes of from about 18 nt to about25, 50, 100 or 250 nt, but 18 nt usually represents sufficient sequencefor unique identification.

Nucleic acids of the invention also include naturally occurring variantsof the nucleotide sequences (e.g., degenerate variants, allelicvariants, etc.). Variants of the nucleic acids of the invention areidentified by hybridization of putative variants with nucleotidesequences disclosed herein, preferably by hybridization under stringentconditions. For example, by using appropriate wash conditions, variantsof the nucleic acids of the invention can be identified where theallelic variant exhibits at most about 25-30% base pair (bp) mismatchesrelative to the selected nucleic acid probe. In general, allelicvariants contain 15-25% bp mismatches, and can contain as little as even5-15%, or 2-5%, or 1-2% bp mismatches, as well as a single bp mismatch.

The invention also encompasses homologs corresponding to the nucleicacids of SEQ ID NO: 5, where the source of homologous genes can be anymammalian species, e.g., primate species, particularly human; rodents,such as rats; canines, felines, bovines, ovines, equines, fish, yeast,nematodes, etc. Between mammalian species, e.g., human and mouse,homologs generally have substantial sequence similarity, e.g., at least75% sequence identity, usually at least 90%, more usually at least 95%between nucleotide sequences. Sequence similarity is calculated based ona reference sequence, which may be a subset of a larger sequence, suchas a conserved motif, coding region, flanking region, etc. A referencesequence will usually be at least about 18 contiguous nt long, moreusually at least about 30 nt long, and may extend to the completesequence that is being compared. Algorithms for sequence analysis areknown in the art, such as gapped BLAST, described in Altschul et al.Nucl. Acids Res. (1997) 25:3389-3402.

The subject nucleic acids can be cDNAs or genomic DNAs, as well asfragments thereof, particularly fragments that encode a biologicallyactive polypeptide and/or are useful in the methods disclosed herein.The term “cDNA” as used herein is intended to include all nucleic acidsthat share the arrangement of sequence elements found in native maturemRNA species, where sequence elements are exons and 3′ and 5′ non-codingregions. Normally mRNA species have contiguous exons, with theintervening introns, when present, being removed by nuclear RNAsplicing, to create a continuous open reading frame encoding apolypeptide of the invention.

A genomic sequence of interest comprises the nucleic acid presentbetween the initiation codon and the stop codon, as defined in thelisted sequences, including all of the introns that are normally presentin a native chromosome. It can further include the 3′ and 5′untranslated regions found in the mature mRNA. It can further includespecific transcriptional and translational regulatory sequences, such aspromoters, enhancers, etc., including about 1 kb, but possibly more, offlanking genomic DNA at either the 5′ and 3′ end of the transcribedregion. The genomic DNA can be isolated as a fragment of 100 kbp orsmaller; and substantially free of flanking chromosomal sequence. Thegenomic DNA flanking the coding region, either 3′ and 5′, or internalregulatory sequences as sometimes found in introns, contains sequencesrequired for proper tissue, stage-specific, or disease-state specificexpression.

The nucleic acid compositions of the subject invention can encode all ora part of the subject polypeptides. Double or single stranded fragmentscan be obtained from the DNA sequence by chemically synthesizingoligonucleotides in accordance with conventional methods, by restrictionenzyme digestion, by PCR amplification, etc. Isolated nucleic acids andnucleic acid fragments of the invention comprise at least about 18,about 50, about 100, to about 200 contiguous nt selected from thenucleic acid sequence as shown in SEQ ID NO: SEQ ID NO: 11, 120 or 122.For the most part, fragments will be of at least 18 nt, usually at least25 nt, and up to at least about 50 contiguous nt in length or more.

Probes specific to the nucleic acid of the invention can be generatedusing the nucleic acid sequence disclosed in SEQ ID NO: 11, or a DNAencoding the polypeptide of SEQ ID NO: 1-10. The probes are preferablyat least about 18 nt, 25nt or more of the corresponding contiguoussequence. The probes can be synthesized chemically or can be generatedfrom longer nucleic acids using restriction enzymes. The probes can belabeled, for example, with a radioactive, biotinylated, or fluorescenttag. Preferably, probes are designed based upon an identifying sequenceof one of the provided sequences. More preferably, probes are designedbased on a contiguous sequence of one of the subject nucleic acids thatremain unmasked following application of a masking program for maskinglow complexity (e.g., BLASTX) to the sequence, i.e., one would select anunmasked region, as indicated by the nucleic acids outside the poly-nstretches of the masked sequence produced by the masking program.

The nucleic acids of the invention are isolated and obtained insubstantial purity, generally as other than an intact chromosome.Usually, the nucleic acids, either as DNA or RNA, will be obtainedsubstantially free of other naturally-occurring nucleic acid sequences,generally being at least about 50%, usually at least about 90% pure andare typically “recombinant,” e.g., flanked by one or more nucleotideswith which: it: is not normally associated on a naturally occurringchromosome.

Retrocyclin encoding nucleic acids can be provided as a linear moleculeor within a circular molecule, and can be provided within autonomouslyreplicating molecules (vectors) or within molecules without replicationsequences. Expression of the nucleic acids can be regulated by their ownor by other regulatory sequences known in the art. The nucleic acids ofthe invention can be introduced into suitable host cells using a varietyof techniques available in the art, such as transferrinpolycation-mediated DNA transfer, transfection with naked orencapsulated nucleic acids, liposome-mediated DNA transfer,intracellular transportation of DNA-coated latex beads, protoplastfusion, viral infection, electroporation, gene gun, calciumphosphate-mediated transfection, and the like.

Expression vectors may be used to introduce a retrocyclin codingsequence into a cell. Such vectors generally have convenient restrictionsites located near the promoter sequence to provide for the insertion ofnucleic acid sequences. Transcription cassettes may be preparedcomprising a transcription initiation region, the target gene orfragment thereof, and a transcriptional termination region. Thetranscription cassettes may be introduced into a variety of vectors,e.g. plasmid; retrovirus, e.g. lentivirus; adenovirus; and the like,where the vectors are able to transiently or stably be maintained in thecells, usually for a period of at least about one day, more usually fora period of at least about several days to several weeks.

The gene or retrocyclin peptide may be introduced into tissues or hostcells by any number of routes, including viral infection,microinjection, or fusion of vesicles. Jet injection may also be usedfor intramuscular administration, as described by Furth et al. (1992)Anal Biochem 205:365-368. The DNA may be coated onto goldmicroparticles, and delivered intradermally by a particle bombardmentdevice, or “gene gun” as described in the literature (see, for example,Tang et al. (1992) Nature 356:152-154), where gold microprojectiles arecoated with the stresscopin or DNA, then bombarded into skin cells.

Methods of Use

Formulations of retrocyclins are administered to a host suffering froman ongoing bacterial or viral infection or who faces exposure to abacterial or viral infection. Antiviral compositions may also utilizeother circular mini-defensins, e.g. RC-101, RTD-1, -2, and -3, alone orin combination with retrocyclin. Administration may be topical,localized or systemic, depending on the specific microorganism.Generally the dosage will be sufficient to decrease the microbial orviral population by at least about 50%, usually by at least 1 log, andmay be by 2 or more logs. The compounds of the present invention areadministered at a dosage that reduces the pathogen population whileminimizing any side-effects. It is contemplated that the compositionwill be obtained and used under the guidance of a physician for in vivouse. Retrocyclins are particularly useful for killing Listeriamonocytogenes and Escherichia coli, and for preventing infection bycertain viruses, particularly enveloped retroviruses, e.g. envelopedretroviruses such as HIV-1, HIV-2, FIV, and the like.

Retrocylins are also useful for in vitro formulations to kill microbes,particularly where one does not wish to introduce quantities ofconventional antibiotics. For example, retrocyclins may be added toanimal and/or human food preparations, or to blood products intended fortransfusion to reduce the risk of consequent bacterial or viralinfection. This may be of particular interest since a common route ofinfection of E. coli and L. monocytogenes is the gastrointestinal tract.Retrocyclins may be included as an additive for in vitro cultures ofcells, to prevent the overgrowth of microbes in tissue culture.

The susceptibility of a particular microbe or virus to killing orinhibition by retrocyclins may be determined by in vitro testing, asdetailed in the experimental section. Typically a culture of the microbeis combined with retrocyclins at varying concentrations for a period oftime sufficient to allow the protein to act, usually ranging from aboutone hour to one day. The viable microbes are then counted, and the levelof killing determined. Two stage radial diffusion assay is a convenientalternative to determining the MIC or minimum inhibitory concentrationof an antimicrobial agent.

Viral pathogens of interest include retroviral pathogens, e.g. HIV-1;HIV-2, HTLV, FIV, SIV, etc. Microbes of interest, but not limited to thefollowing, include: Citrobacter sp.; Enterobacter sp.; Escherichia sp.,e.g. E. coli; Klebsiella sp.; Morganella sp.; Proteus sp.; Providenciasp.; Salmonella sp., e.g. S. typhi, S. typhimurium; Serratia sp.;Shigella sp.; Pseudomonas sp., e.g. P. aeruginosa; Yersinia sp., e.g. Y.pestis, Y. pseudotuberculosis, Y enterocolitica; Franciscella sp.;Pasturella sp.; Vibrio sp., e.g. V. cholerae, V. parahemolyticus;Campylobacter sp., e.g. C. jejuni; Haemophilus sp., e.g. H. influenzae,H. ducreyi; Bordetella sp., e.g. B. pertussis, B. bronchiseptica, B.parapertussis; Brucella sp., Neisseria sp., e.g. N. gonorrhoeae, N.meningitidis, etc. Other bacteria of interest include Legionella sp.,e.g. L. pneumophila; Listeria sp., e.g. L. monocytogenes; Staphylococcussp., e.g. S. aureusMycoplasma sp., e.g. M. hominis, M. pneumoniae;Mycobacterium sp., e.g. M. tuberculosis, M. leprae; Treponema sp., e.g.T. pallidum; Borrelia sp., e.g. B. burgdorferi; Leptospirae sp.;Rickettsia sp., e.g. R. rickettsii, R. typhi; Chlamydia sp., e.g. C.trachomatis, C. pneumoniae, C. psittaci;Helicobacter sp. e.g. H. pylon,etc.

Various methods for administration may be employed. For the preventionof HIV infection, administration to mucosal surfaces is of particularinterest, e.g. vaginal, rectal, etc. The polypeptide formulation may begiven orally, or may be injected intravascularly, subcutaneously,peritoneally, by aerosol, opthalmically, intra-bladder, topically, etc.For example, methods of administration by inhalation are well-known inthe art. The dosage of the therapeutic formulation will vary widely,depending on the specific retrocyclin or demi-defensin to beadministered, the nature of the disease, the frequency ofadministration, the manner of administration, the clearance of the agentfrom the host, and the like. The initial dose may be larger, followed bysmaller maintenance doses. The dose may be administered as infrequentlyas weekly or biweekly, or fractionated into smaller doses andadministered once or several times daily, semi-weekly, etc. to maintainan effective dosage level. In many cases, oral administration willrequire a higher dose than if administered intravenously. The amidebonds, as well as the amino and carboxy termini, may be modified forgreater stability on oral administration.

Formulations

The compounds of this invention can be incorporated into a variety offormulations for therapeutic administration. More particularly, thecompounds of the present invention can be formulated into pharmaceuticalcompositions by combination with appropriate, pharmaceuticallyacceptable carriers or diluents, and may be formulated into preparationsin solid, semi-solid, liquid or gaseous forms, such as tablets,capsules, powders, granules, ointments, solutions, suppositories,injections, inhalants, gels, microspheres, lotions, and aerosols. Assuch, administration of the compounds can be achieved in various ways,including oral, vaginal, buccal, rectal, parenteral, intraperitoneal,intradermal, transdermal, intratracheal, etc., administration. Theretrocyclins may be systemic after administration or may be localized bythe use of an implant or other formulation that acts to retain theactive dose at the site of implantation.

The compounds of the present invention can be administered alone, incombination with each other, or they can be used in combination withother known compounds (e.g., perforin, anti-inflammatory agents,antibiotics, etc.) In pharmaceutical dosage forms, the compounds may beadministered in the form of their pharmaceutically acceptable salts. Thefollowing methods and excipients are merely exemplary and are in no waylimiting.

For oral preparations, the compounds can be used alone or in combinationwith appropriate additives to make tablets, powders, granules orcapsules, for example, with conventional additives, such as lactose,mannitol, corn starch or potato starch; with binders, such ascrystalline cellulose, cellulose derivatives, acacia, corn starch orgelatins; with disintegrators, such as corn starch, potato starch orsodium carboxymethylcellulose; with lubricants, such as talc ormagnesium stearate; and if desired, with diluents, buffering agents,moistening agents, preservatives and flavoring agents.

The compounds can be formulated into preparations for injections bydissolving, suspending or emulsifying them in an aqueous or nonaqueoussolvent, such as vegetable or other similar oils, synthetic aliphaticacid glycerides, esters of higher aliphatic acids or propylene glycol;and if desired, with conventional additives such as solubilizers,isotonic agents, suspending agents, emulsifying agents, stabilizers andpreservatives.

The compounds can be utilized in aerosol formulation to be administeredvia inhalation. The compounds of the present invention can be formulatedinto pressurized acceptable propellants such as dichlorodifluoromethane,propane, nitrogen and the like.

The compounds can be used as lotions, for example to prevent infectionof burns, by formulation with conventional additives such assolubilizers, isotonic agents, suspending agents, emulsifying agents,stabilizers and preservatives.

Furthermore, the compounds can be made into suppositories by mixing witha variety of bases such as emulsifying bases or water-soluble bases. Thecompounds of the present invention can be administered rectally via asuppository. The suppository can include vehicles such as cocoa butter,carbowaxes and polyethylene glycols, which melt at body temperature, yetare solidified at room temperature.

Unit dosage forms for oral, vaginal or rectal administration such assyrups, elixirs, and suspensions may be provided wherein each dosageunit, for example, teaspoonful, tablespoonful, tablet or suppository,contains a predetermined amount of the composition containing one ormore compounds of the present invention. Similarly, unit dosage formsfor injection or intravenous administration may comprise the compound ofthe present invention in a composition as a solution in sterile water,normal saline or another pharmaceutically acceptable carrier.

Implants for sustained release formulations are well-known in the art.Implants are formulated as microspheres, slabs, etc. with biodegradableor non-biodegradable polymers. For example, polymers of lactic acidand/or glycolic acid form an erodible polymer that is well-tolerated bythe host. The implant containing retrocyclins is placed in proximity tothe site of infection, so that the local concentration of active agentis increased relative to the rest of the body.

The term “unit dosage form”, as used herein, refers to physicallydiscrete units suitable as unitary dosages for human and animalsubjects, each unit containing a predetermined quantity of compounds ofthe present invention calculated in an amount sufficient to produce thedesired effect in association with a pharmaceutically acceptablediluent, carrier or vehicle. The specifications for the unit dosageforms of the present invention depend on the particular compoundemployed and the effect to be achieved, and the pharmacodynamicsassociated with the compound in the host.

The pharmaceutically acceptable excipients, such as vehicles, adjuvants,carriers or diluents, are readily available to the public. Moreover,pharmaceutically acceptable auxiliary substances, such as pH adjustingand buffering agents, tonicity adjusting agents, stabilizers, wettingagents and the like, are readily available to the public.

Typical dosages for systemic administration range from 0.1 μg to 100milligrams per kg weight of subject per administration. A typical dosagemay be one tablet taken from two to six times daily, or one time-releasecapsule or tablet taken once a day and containing a proportionallyhigher content of active ingredient. The time-release effect may beobtained by capsule materials that dissolve at different pH values, bycapsules that release slowly by osmotic pressure, or by any other knownmeans of controlled release.

Those of skill will readily appreciate that dose levels can vary as afunction of the specific compound, the severity of the symptoms and thesusceptibility of the subject to side effects. Some of the specificcompounds are more potent than others. Preferred dosages for a givencompound are readily determinable by those of skill in the art by avariety of means. A preferred means is to measure the physiologicalpotency of a given compound.

The use of liposomes as a delivery vehicle is one method of interest.The liposomes fuse with the cells of the target site and deliver thecontents of the lumen intracellularly. The liposomes are maintained incontact with the cells for sufficient time for fusion, using variousmeans to maintain contact, such as isolation, binding agents, and thelike. In one aspect of the invention, liposomes are designed to beaerosolized for pulmonary administration. Liposomes may be prepared withpurified proteins or peptides that mediate fusion of membranes, such asSendai virus or influenza virus, etc. The lipids may be any usefulcombination of known liposome forming lipids, including cationic orzwitterionic lipids, such as phosphatidylcholine. The remaining lipidwill be normally be neutral or acidic lipids, such as cholesterol,phosphatidyl serine, phosphatidyl glycerol, and the like.

For preparing the liposomes, the procedure described by Kato et al.(1991) J. Biol. Chem. 266:3361 may be used. Briefly, the lipids andlumen composition containing peptides are combined in an appropriateaqueous medium, conveniently a saline medium where the total solids willbe in the range of about 1-10 weight percent. After intense agitationfor short periods of time, from about 5-60 sec., the tube is placed in awarm water bath, from about 25-40° C. and this cycle repeated from about5-10 times. The composition is then sonicated for a convenient period oftime, generally from about 1-10 sec. and may be further agitated byvortexing. The volume is then expanded by adding aqueous medium,generally increasing the volume by about from 1-2 fold, followed byshaking and cooling. This method allows for the incorporation into thelumen of high molecular weight molecules.

Formulations With Other Active Agents

For use in the subject methods, retrocyclins may be formulated withother pharmaceutically active agents, particularly other antimicrobialagents. Other agents of interest include a wide variety of antibiotics,as known in the art. Classes of antibiotics include penicillins, e.g.penicillin G, penicillin V, methicillin, oxacillin, carbenicillin,nafcillin, ampicillin, etc.; penicillins in combination with β-lactamaseinhibitors, cephalosporins, e.g. cefaclor, cefazolin, cefuroxime,moxalactam, etc.; carbapenems; monobactams; aminoglycosides;tetracyclines; macrolides; lincomycins; polymyxins; sulfonamides;quinolones; cloramphenical; metronidazole; spectinomycin; trimethoprim;vancomycin; etc.

Cytokines may also be included in a retrocyclin formulation, e.g.interferon γ, tumor necrosis factor α, interleukin 12, etc.

Antiviral agents, e.g. acyclovir, gancyclovir, etc., and other circularmini-defensins (theta defensins) may also be included in retrocyclinformulations.

EXPERIMENTAL

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the subject invention, and are not intended to limit thescope of what is regarded as the invention. Efforts have been made toensure accuracy with respect to the numbers used (e.g. amounts,temperature, concentrations, etc.) but some experimental errors anddeviations should be allowed for. Unless otherwise indicated, parts areparts by weight, molecular weight is average molecular weight,temperature is in degrees centigrade; and pressure is at or nearatmospheric.

Sexual and mother-to-neonate (vertical) transmission through mucosalsurfaces have been the most common routes of HIV-1 spread throughout theworld. Although much attention has been focused on vaccine developmentfor HIV-1, progress has been slow and there is an urgent need to findalternative approaches to prevent infections caused by HIV-1.Self-applied prophylactic agents to prevent mucosal, particularlyvaginal or rectal, transmission of HIV-1 have the advantage ofempowering vulnerable receptive partners to take effective measures fortheir own protection. In a search for novel compounds active againstHIV-1, it was discovered that certain antimicrobial peptides, thecircular minidefensins from the rhesus macaque, could inhibit HIV-1replication. This prompted an investigation as to whether humans producecircular minidefensins. Although there is no evidence that theseproteins are produced in humans, clearly some primate ancestors oncemade retrocyclin, because it continues to exist in contemporary humansas an expressed pseudogene.

After discovering an mRNA molecule in human bone marrow that was highlyhomologous to rhesus circular minidefensins (88.9% identity at thenucleotide level), solid phase peptide synthesis was used to create thepeptide (“retrocyclin”) whose sequence it encoded. Retrocyclin belongsto the θ defensin subfamily (also referred to as cyclic minidefensins).The antimicrobial properties of retrocyclin resemble those of rhesusθ-defensins. However, retrocyclin is highly effective in preventing theinfection of CD4⁺ cells by X4 and R5 strains of HIV-1 in vitro.

Example 1 Circular Minidefensins can Block HIV-1 Replication

It is shown herein that retrocyclin, a circular minidefensin, ispotently active against both X4 and R5 strains of HIV-1. The initialdescriptions of circular minidefensins came from studies of Macacamulatta, the rhesus macaque monkey. The first such peptide, RTD-1, wascalled a rhesus theta defensin (RTD), which are also referred to as“cyclic minidefensins”. The peptides encoded by the mRNA precursors maybe referred to as “demidefensins”.

RTD-2 and RTD-3, which was isolated from the bone marrow of rhesusmonkeys, are circular, 18 amino acid peptides that contained threeintramolecular disulfide bonds. They are similar to RTD-1,the circular(θ) defensin previously described by Tang et a. However, whereas the 18residues of RTD-1 represent spliced 9 amino acid fragments derived fromtwo different minidefensin precursors, RTD-2 and -3 comprise tandem 9residue repeats derived from a single RTD-1 precursor. Thus, circularminidefensins are processed by a novel post-translational system thatcan generates a degree of effector molecule diversity without requiringcommensurate genome expansion.

Retrocyclin and the other circular minidefensins we prepared weresynthesized, folded, circularized and purified essentially. Theantiviral activities of RTD-1, RTD-2 and RTD-3 are shown in FIG. 1. Forthese studies, the X4 HIV-1 strain IIIB was utilized.

Immortalized CD4⁺H9 cells, which are permissive for infection with thisstrain, were maintained in RPMI supplemented with 10% heat-inactivatedfetal calf serum (FCS), 10 mM HEPES, 2 mM glutamine, 100 U ofpenicillin/ml, and 10 μg of streptomycin/ml (R10 media). Cells(2.5×10⁵/100 μl) were incubated with virus (multiplicity of infection(MOI)=10⁻²) in the presence or absence of 20 μg/ml RTD-1, RTD-2 or RTD-3for 3 hrs at 37° C./5% CO₂. The cells were washed in R10 media, seededin 48-well tissue culture plates in 1 ml R10 media, and incubated at 37°C./5% CO₂ for 9 days. Aliquots of cell supernatant were removed at thespecified time points and analyzed by a sensitive ELISA (DuPont NEN)that quantitates p24 antigen of HIV-1. The three circular rhesusminidefensins were similarly active, inhibiting HIV-1 by 100-1000 foldby 9 days post-inoculation (note the logarithmic scale).

Example 2 Identification and Structural Characterization of Retrocyclin

To search for human circular minidefensins, two primers were preparedbased on the monkey minidefensin cDNA sequences (GenBank AF 184156,184157, 184158). When PCR was performed on Marathon-Ready human bonemarrow cDNA (Clontech, Palo Alto, Calif.), a 264 bp amplified productwas recovered. To obtain its 3′ and 5′ side sequences, Marathon-Readyhuman bone marrow cDNA was amplified using a 3′-RACE kit (Gibco BRL,Gaithersburg, Md.) and 5′-RACE kit from Boehringer Mannheim(Indianapolis, Ind.).

At the nucleotide level, this product (retrocyclin) was 89% identical tothe demidefensin precursors of rhesus RTD-1 (called precursors 1 a and 1b). FIG. 2 shows the peptide sequences of demidefensin 1 andpreproretrocyclin. Residues incorporated into the mature circularminidefensins are boxed and all stop codons are represented by solidcircles. Although a stop codon within the human transcript's signalsequence should abort translation, the otherwise high conservation ofrhesus and human mRNA's suggested that humans may have acquired thismutation relatively recently in primate evolution.

Three orangutan retrocyclin genes have been sequences. One of theseclimes has the silencing stop codon in the signal sequence and thereforeresembles human retrocyclin. The other two orangutan genes appear to befunctional, i.e. when translated they would produce demi-defensins, theprecursors of cyclic minidefensins.

Human leukocytes were examined for the presence of retrocyclin orsimilar peptides, but, as expected from the presence of the signalsequence's stop codon, none was found: Thus synthetic retrocyclinrepresents the circular minidefensin that would have formed: a) if thesignal sequence mutation were absent, and b) if the precursor underwenthomologous pairing so that its boxed residues (see FIG. 2) formed bothhalves of the circular molecule.

In phylogenetic studies of the retrocyclin demidefensin gene, thepremature stop codon in the signal sequence was found to be present infour anthropoid species (humans, gorillas, chimpanzees, pygmychimpanzees) and not present in the genes of a fifth (orangutangs). Thedemidefensin gene also appears intact (i.e., no premature stop codon) inthe two catarrhine (Old World Monkey) species examined to date, Macacamulatta and Macaca nemestrina. These findings suggest that nativeretrocyclin peptides were last produced by a primate ancestor of humansand other anthropoids that lived between 6 and 15 million years ago(mya). This is between the time that orangutang and human lineagediverged (15 mya) and before the divergence of the chimpanzee and humanlineages (6 mya). These ongoing studies of primate phylogeny may yieldsequence information about additional cyclic minidefensins whose nativecounterparts are extinct.

Retrocyclin synthesis. Peptides were synthesized at a 0.25 mmol scalewith a Perkin-Elmer ABI431A Synthesizer, using pre-derivatizedpolyethylene glycol polystyrene arginine resin (PerSeptive Biosystems,Framingham, Mass.), FastMoc™ chemistry, and double coupling for allresidues. The crude peptide was reduced under nitrogen, for 15 hours at50° C. with excess dithiothreitol in 6 M guanidine.HCl, 0.2 M Tris.HCland 0.2 mM EDTA (pH 8.2). The reaction was stopped with glacial aceticacid (final concentration, 5%) and the reduced peptide was stored undernitrogen until purified by RP-HPLC. After this step, the peptideappeared homogeneous and its mass (1942.5, by MALDI-TOF MS) agreed wellwith its theoretical mass. The reduced peptide (0.1 mg/ml) was oxidized,cyclized and purified essentially as described by Tang et al., supra.The MALDI-TOF MS mass of retrocyclin (1918.5 Da) agreed well with itsexpected mass. CD spectra were obtained at 25° C. from an AVIV 62DSspectropolarimeter (AVIV, Lakewood, N.J.).

RTD-1 and retrocyclin have very similar CD spectra, with largely β-sheetstructures stabilized by disulfide linkages and connected by turns (FIG.3A). Antimicrobial peptides with similar spectra include tachyplesins,protegrins, and circularized defensins. FIG. 3B, a backbone ribbon modelof retrocyclin, was made by templating its sequence on the structure ofprotegrin PG-1 and cyclizing it. The resulting structure was annealed bymolecular dynamics and energy minimized. FIG. 3D is a cartoon version ofFIG. 3B, designed primarily to show the placement of the cysteine andarginine molecules. FIG. 3C is a similar cartoon of rhesus RTD-1.

Retrocyclin is a selectively salt-insensitive antibacterial peptide. Theeffects of NaCl on the antimicrobial activity of retrocyclin and RTD-1,from two-stage radial diffusion assays, are compared in FIG. 4. Thepeptides showed very similar behavior. Under low salt conditions, bothpeptides were highly effective (minimal inhibitory concentration (MIC)<3μg/ml) against all four test organisms: Pseudomonas aeruginosa,Escherichia coli, Listeria monocytogenes and Staphylococcus aureus.Their strong activity against E. coli and L. monocytogenes persisted inphysiological (100 mM) NaCl, and even hypersalinity (175 mM NaCl) wasonly modestly inhibitory. In contrast, neither peptide was effective(MIC>50 μg/ml) against S. aureus or P. aeruginosa in physiological orhigh salt concentrations. Retrocyclin's activity is likely to bepreserved in the ionic concentration of the vaginal mucosa.

Retrocyclin potently inhibits HIV-1 replication of R5 and X4 viruses.The antiretroviral properties of retrocyclin are shown in FIG. 5. EitherHIV-1-permissive H9 cells were used as targets, or primary CD4⁺lymphocytes from HIV-1-seronegative donors generated from freshlypurified peripheral blood mononuclear cells (PBMC) stimulated with aCD3-CD8 bispecific monoclonal antibody. After approximately 7 days, when98% of these cells co-expressed CD3 and CD4, they were infected withHIV-1 with or without retrocyclin or other test peptide. These cellswere maintained in RPMI containing 10% FCS supplemented with 2 mMglutamine, 100 U of penicillin/ml, 10 μg of streptomycin/ml, and 50 U ofinterleukin 2/ml (R10-50 media).

Retrocyclin (10-20 μg/ml) afforded complete suppression of viralreplication to CD4⁺-selected PBMC challenged with two different strainsof HIV-1: IIIB (an X4 strain) and JR-CSF (an R5 strain), or H9 human Tcells challenged with IIIB. Note that the concentration of p24 antigenis presented on a log-scale and that the rhesus circular minidefensins,RTD-1 (FIG. 5 and FIG. 1) and RTD-2 and RTD-3 were protective to alesser extent than retrocyclin. Additionally, the antiretroviralactivities of T140 (20 μg/ml; FIG. 5) and T22, analogs of polyphemusinsfrom horseshoe crabs that were previously shown to protect against X4,but not R5, infections, were confirmed in the present study.Microbicides, such as retrocyclin, that target both X4 and R5 viruses bemore effective than agents that preferentially inhibit viruses of asingle tropism.

Examining the effect of adding retrocyclin at various times pre- andpost-HIV-1 infection.

To determine if retrocyclin is effective against HIV-1 when addedpost-infection, we either: 1) added retrocyclin at the time of HIV-1infection, then washed away the peptide, or 2) added retrocyclin atvarious times post-infection. Primary CD4⁺PBMC were incubated withHIV-IIIB (FIG. 6) or HIV-JR-CSF for 3 hours in the presence or absenceof 20 μg/ml retrocyclin. The cells were subsequently washed in media,and incubated an additional 9 days. Retrocyclin (20 μg/ml) was addedback to some of the cultures at time points specified in FIG. 6 andinfection was monitored by p24 ELISA as previously described. Althoughretrocyclin was most active when administered at the time of infection,and when present in culture throughout the 9 day incubation, retrocyclinadministered as late as 24 hours after initial infection still reducedthe p24 concentration by nearly 1000-fold.

Cyclization and oxidation are necessary for retrocyclin's antiviralactivity. Mature retrocyclin was prepared by a three-step process. Itstwo intermediate forms, as well as the final retrocyclin product weretested in our standard assay of HIV-1 infectivity: p24 ELISA of HIV-IIIBinfection of H9 cells (FIG. 7). Intermediate 1 (open triangles) is thelinearized retrocyclin octadecapeptide with 6 reduced cysteine thiolgroups. Intermediate 2 (closed triangles) is a noncyclic b-hairpinoctadecapeptide with 3 intramolecular cystine disulfide bonds.Retrocyclin (open squares), is a cyclized octadecapeptide with 3disulfide bonds. Note that only the “mature” form of retrocyclin wasantiviral, compared to control (no retrocyclin, open circles). Unlikeretrocyclin, the linearized octadecapeptide was highly cytotoxic, asmeasured by trypan blue exclusion, and treated cells did not survivepast 6 days.

Retrocyclin is not cytotoxic. Cytotoxicity determinations were made witha Cell Proliferation Kit from Boehringer Mannheim used according to themanufacturer's instructions. The procedure measures the reduction of theyellowish MTT molecule (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) to a dark blue formazan. Retrocyclin exhibitedlittle to no cytotoxicity against H9 cells (FIG. 8) and ME-180 cervicalcarcinoma cells at 100 μg/ml, a concentration that is far higher thanthe concentrations required for complete protection against HIV-1infection (10 μg/ml). Additionally, neither 20 μg/ml retrocyclin norRC-101 were cytotoxic to HIV-1-infected H9 cells and CD4⁺PBMC asmeasured by trypan-blue exclusion (Table 1). Retrocyclin was nothemolytic for human erythrocytes.

TABLE 1 Cytotoxicity of 20 μg/ml peptide against H9 cells and CD4⁺ PBMC(peripheral blood mononuclear cells) as measured by Trypan blueexclusion. Cells; virus no peptide* Retrocyclin CD4⁺ PBMC; no virus 1.070.98 H9; IIIB 0.78 1.20 CD4⁺ PBMC; IIIB 1.71 1.68 CD4⁺ PBMC; JR-CSF 0.901.58 *Values expressed as the average number of cells × 10⁶/ml for 2-3experiments. N.D. = no data.

Construction and characterization of retrocyclin congeners. To date, wehave constructed over a dozen congeners of retrocyclin, “RC-101”,“RC-102”, “RC-103”, etc. and have used them to commence astructure-activity analysis of the retrocyclin's antiviral andantimicrobial effects. These peptides, whose sequences are shown inTable 2, were synthesized, oxidized, cyclized, and purified as describedabove for retrocyclin. RC-101 was prepared because retrocyclin, acircular peptide without free N-terminal or side-chain amine groups, isnot well suited for fluorescent-conjugation. RC-101 is identical insequence to retrocyclin (RC-100) except for the presence of an Arg₉→Lys₉substitution. This modification preserves the net cationic charge of thepeptide and provides an available epsilon-amino group in lysine's sidechain. Importantly, RC-101 was as active as retrocyclin in protectingcells from infection by HIV-1 (FIG. 11), indicating that substitutionsin the primary sequence of retrocyclins can be made without losinganti-retroviral activity. The labeling of RC-101 with amine-reactiveprobes will be described in a later section.

Five additional analogues (RC 110-114) have been synthesized, cyclizedand purified. RC-110 (Inverso-enantioretrocyclin), a cyclic peptidecomposed exclusively of D amino acids, has a sequence that is identicalto retrocyclin, but with its residues placed in reverse order.

The ability of RC100 and several analogues described in Table 2 toprotect cells from infection by X4 (HIV-IIIB) and R5 (JR-CSF) strains ofHIV-1 is shown in FIG. 9. The structure of retrocyclin itself is shownin FIG. 10, with its residues numbered to correspond to Table 2.

The p24 assay results shown in FIG. 9 are on a logarithmic scale. Ahorizontal reference line that passes through 10⁰ on the ordinate scalecorresponds to 1 pg/ml of p24 antigen. Results from 3 experiments (eachperformed with PBMC from a different donor) are shown. Retrocyclin wasuniformly protective against both strains of HIV-1 in all of theexperiments. Most of the mono-tyrosine substituted amino acid congenersof retrocyclin (RC102-RC-103, RC-105, RC-106, and RC-108) were eitherinactive or only modestly active in inhibiting HIV-1 infection by StrainIIIB. In contrast, RC-102, RC-103 and RC-104 showed considerable abilityto protect cells from infection by the JR-CSF strain (R5).

These results allow some hypotheses about the mechanism of action ofretrocyclins to be formulated. Because RC-112 (enantioRetrocyclin) wasrelatively ineffective, chiral interactions between retrocyclin and oneor more receptors on the cell and/or virus surface are likely toparticipate in the protective mechanism. Since certain analogues(RC-102, RC-103 and perhaps RC104) manifested substantial activityagainst the R5 strain but were relatively ineffective against the X4strain, the mechanisms whereby retrocyclin inhibits these strains arenot identical. The lack of efficacy of RC-106, RC-107 and RC-108 (eachcontaining a tyrosine for arginine replacemene) suggests that ionicinteractions involving the positively charged arginine residues inposition 4, 9, and 13 of retrocyclin (see the model in FIG. 10) withoppositely charged groups (e.g., phosphate) on the surface of the targetcell or HIV-1 virion also participate in the process. In preliminarysurface plasmon resonance (SPR) experiments, we have observed thatretrocyclin with high affinity to certain sphingolipids (e.g.,galactosylceramide) that are present in cell-surface rafts, and havebeen implicated in the cellular uptake of HIV-1 virions.

TABLE 2 Primary amino acid sequence of selected retrocyclin congeners.Name Avg. SEQ ID NO: Peptide (or comment) (Da) MW Amino acid sequence 1RC-100* Retrocyclin 1918.4 GICRCICGRGICRCICGR 2 RC-101 R₉K-Retrocyclin1890.4 GICRCICGKGICRCICGR 3 RC-102 I₆Y-Retrocyclin 1968.5GICRCYCGRGICRCICGR 4 RC-103 I₁₅Y-Retrocyclin 1968.5 GICRCICGRGICRCYCGR 5RC-104 I₂Y-Retrocyclin 1968.5 GYCRCICGRGICRCICGR 6 RC-105I₁₁Y-Retrocyclin 1968.5 GICRCICGRGYCRCICGR 7 RC-106 R₄Y-Retrocyclin1925.4 GICYCICGRGICRCICGR 8 RC-107 R₉Y-Retrocyclin 1925.4GICICICGYGICRCICGR 9 RC-108 R₁₃Y-Retrocyclin 1925.4 GICICICGRGICYCICGR10 RC-109** GICICICGRGICRCICGY 19 RC-110 Inverso-enantio- 1918.4RGCICRCIGRGCTCRCIG Retrocyclin (ALL D) 20 RC-111 Inverso-retrocyclin1918.4 RGCICRCIGRGCICRCIG 21 RC-112 enantio-retrocyclin 1918.4GICRCICGRGICRCICGR (all D) 22 RC-113 enantio-RC-101 1890.4GICRCICGKGICRCICGR (all D) 23 RC-114 RC-101/103 1940.4GICRCICGKGICRCYCGR hybrid With the exception of RC-109, all of the abovepeptides are cyclic. *RC-100 is a synonym for retrocyclin, RC-111(inverso-retrocyclin) is composed of L-amino acids; RC-110, 112 and 113are composed exclusively of D-amino acids. RC-109 failed to cyclize, andhas not been tested further.

!

Retrocyclin does not directly inactivate HIV-1. To determine ifretrocyclin directly inactivated HIV-1 virions, HIV-IIIB (MOI 10⁻²) wasincubated with 2 μg/ml, 20 μg/ml, or 200 μg/ml retrocyclin for 30 min atroom temperature in R10 media. The mixture was diluted 190-fold in R10media, to dilute retrocyclin below its effective antiviralconcentrations (no significant antiviral activity at <2 μg/ml; n=5), andused to infect 5×10⁵ H9 CD4⁺ cells. Viral replication was measured bycollecting supernatant for 9 days at 3 day intervals to quantify HIV-1p24 antigen by ELISA (FIG. 12). HIV titer was not reduced with thehighest concentration (200 μg/ml) of retrocyclin, demonstrating thatretrocyclin does not target the virion directly. In this respect, theactions of retrocyclin are different from the direct inactivation ofherpes simplex virus previously observed with human and rabbitα-defensins.

Retrocyclin binds to T1 cells. Since retrocyclin does not directlyinactivate HIV-1 virions, the ability of retrocyclin to interact with acellular target was determined, using RC-101, a Arg₉→Lys₉ congener ofretrocyclin that retained the antiretroviral activity of the parentmolecule. RC-101 was conjugated to the amine-reactive fluorescent dye,BODIPY-FL (Molecular Probes), according to the manufacturer's protocol.The conjugate (RC-101_(BODIPY-FL)) was purified by reverse-phase HPLCand resuspended in 0.01% acetic acid at up to 240 μg/ml.RC-101_(BODIPY-FL) (20 μg/ml) was incubated with 2.5×10⁵ CD4⁺-selectedPBMC cells for 15 min at room temperature, washed once in fresh R10-50media. Specimens were imaged on a Leica TCS-SP Confocal Microscope(Heidelberg, Germany) equipped with an argon laser for excitation ofBODIPY-FL and phycoerythrin (PE). Images were collected with LeicaConfocal Software. RC-101_(BODIPY-FL) bound to the cell membrane, mostlyin patches. Patching (“microaggregation”) has been reported to occurwith hormone-occupied epidermal growth factor receptors (95), and“rafts” are involved in signaling through the confinement of chemokinereceptors to discrete regions of the cell membrane. RC-101_(BODIPY-FL)colocalizes with phycoerythrin (PE)-labeled monoclonal antibodiesdirected against CXCR4, CCR5 and CD4, but does not with PE-labeledisotype control antibodies. Thus, retrocyclin aggregates in the same“rafts” as the receptor and coreceptors for HIV-1. In addition,RC-101_(BODIPY-FL) aggregated in patches where CD4, CXCR4 and CCR5levels were weak or absent

A flow cytometry experiment was performed to examine binding ofBODIPY-labeled RC-101. T1 cells were incubated for 1 hr at 37° C.±20μg/ml RC-101_(BODIPY-FL), washed with R10 media, and fixed in 2%paraformaldehyde/PBS. Cells were analyzed by fluorescence-activated cellsorting (FACS) on a Becton-Dickinson FACScan. Live cells (10⁴ events)were gated and analyzed by CellQuest. Two peaks were present, which mayrepresent non-specific and specific cellular binding

Retrocyclin inhibits HIV replication at an early step (reversetranscription or before). To determine whether retrocyclin blocked theformation of proviral DNA in HIV-JR-CSF-inoculated CD4⁺-selected PBMC,quantitative real time PCR was performed. This method is more sensitivethan measuring p24 release and can detect infection even when p24 valuesmay be affected by virus carried over from the original inoculum.CD4⁺-selected PBMC (10⁶ cells) were incubated in 250 μl at 37° C./5% CO₂for 3 hr with either HIV-1 strain JR-CSF (MOI=0.1), heat-inactivatedvirus (background control), JR-CSF+20 μg/ml retrocyclin or JR-CSF+20 μgRC-101. Cells were washed and resuspended in 1 ml R10-50 media, andincubated for an additional 9 hours. Following incubation, cells werepelleted at 300× g, removed of overlying supernatant, and stored at −80°C. until analyzed by real time PCR. Retrocyclin and RC-101 inhibited theformation of HIV-1 proviral DNA (FIG. 13), indicating that retrocyclinacts early, either to inhibit reverse transcription or preceding events.

Some retrocyclins are slightly active against herpes simplex virus(HSV). To determine if the activity of retrocyclin against HIV-1 wasspecific or representative of a more global antiviral effect, it wastested its ability to prevent HSV infection in vitro. A quantitativemicroplate assay was used to screen retrocyclin andretrocyclin-congeners for their ability to inactivate HSV type 1 (HSV-1)and HSV-2. The assay utilized small amounts of peptide andsimultaneously evaluated for peptide-induced cytotoxicity. In brief,final peptide concentrations of 2-50 μg/ml were incubated with virusstocks for 2 hrs and added directly to ME-180 human cervical carcinomacells. Cultures were incubated at 37° C. for 72 hrs and cytotoxicity wasmeasured using a 3-(4,5-dimethylthiazon-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) cell proliferation kit (Boehringer-Mannheim, Germany).Calculations to assess antiviral activity and compute “percentprotection” are delineated in (90). Retrocyclin afforded modestprotection against HSV-2, but not HSV-1 (FIG. 14). In contrast, RC-102and RC-103 were less antiviral than retrocyclin. However, RC-101 wasmodestly protective against HSV-1 and nearly completely protectiveagainst HSV-2. The substitution of Arg₉→Lys₉ produced a retrocyclincongener that retained activity against HIV-1, bacteria and fungi, andwas more active against HSV.

Example 3 Sequences of Retrocyclins

We have obtained samples of DNA from the 22 primate species listed belowin the Table. The first column shows the phylogenetic group, and has thefollowing key: E. Great apes/humans; C: Old World Monkeys; B: New WorldMonkeys, D. Lesser Apes; A. Column 3, showing “Genetic distance” fromHomo sapiens, is expressed in %. These values come from study of gammaglobin DNA (Page, SL., Chiu C, and Goodman, M. Molecular phylogeny ofOld World Monkeys (Cercopithecidae) as inferred from gamma-globin DNAsequences. Molecular Phylogenetics and Evolution. 1999. 13:348-359.)Column four shows the sources of DNA samples: 1, the Coriell Institute;and 2, the “Frozen Zoo” collection of the San Diego Zoo. We haveperformed RT-PCR on all of these samples with primers based on thesequence of the human retrocyclin gene. “Pending” means that the studiesare still undergoing confirmation.

All of the great apes (Group E) have a retrocyclin gene or pseudogene.In the gorilla and chimpanzee the gene was inactivated by the identicalsignal sequence stop codon mutation found in the human retrocyclinpseudogene. A Sumatran orangutan DNA sample obtained from the CoriellInstitute contained two retrocyclin genes. One gene contained the signalsequence mutation and the other appeared intact. Additional orangutanDNA samples have been obtained from the “Frozen Zoo” (described below)and are being characterized. None of the 6 New World monkeys (Group B)or 5 the prosimian species (Group A) had a retrocyclin gene.

TABLE 3 Genetic DNA Retrocyclin Retrocyclin Group Species DistanceSource Gene Gene Status E Homo sapiens 0.0 1 Present inactivate E Pantroglodytes (chimpanzee) 1.6 1 Present inactivate E Pan paniscus(Bonobo) 1.7 1 Present inactivate E Gorilla gorilla (Lowland gorilla)1.9 1 Present inactivate E Pongo pygmaeus pyg. (Borneo orangutan) 3.1 2pending pending E Pongo pygmaeus abeli (Sumatra orangutan) 3.1 1,2Present intact/inactivate D Hylobates syndactylus (siamang) 4.2 2Present pending C Macaca mulatta (Rhesus macaque) 6.1 1 Present intact CMacaca nemestrina (pigtail macaque) 6.4 1 Present intact C Theropithecusgelada (Gerlada baboon) 6.3 2 pending pending C Colobus guereza kiku.(Kikuyu colobus) 6.8 2 Present pending B Alouatta seniculus (Red Howlermonkey) 12.7  2 Absent n.a. B Callicebus moloch (Titi monkey) — 2 Absentn.a. B Pithecia pithecia (white-faced Saki) — 2 Absent n.a. B Sanguinusfuscicollis (Saddleback tamarin) — 2 Absent n.a. B Callithrix pygmaea(Pygmy marmoset) — 2 Absent n.a. B Ateles geoffroyi (Black handed spidermky) 11.7  1 Absent n.a. A Sanguinus labiatus (Red-bellied tamarin) — 1Absent n.a. A Lemur catta (Ring-tailed lemur) ˜21.3  1 Absent n.a. AVarecia variegata ruber (lemur) — 2 Absent n.a. A Otolemurcrassicaudatus (galago) 28.1 2 Absent n.a. A Nycticebus coucang (Bengalslow loris) — 2 Absent n.a.

Our inferences from these results are: a) The α-defensin precursor ofthe retrocyclin gene appeared after the Old World (B) and New World (C)monkey lineages separated. The retrocyclin gene appeared in Old WorldMonkeys (B) and persisted until the orangutan lineage split from thelineage of Gorillas/Chimpanzees/Humans, some 15 million years ago.

In a recent search of the NCBI human genome data base, two additionalhuman retrocyclin genes were identified. Each gene contains a silencingmutation in its signal sequence. Two of the three retrocyclin genesencodes the same nonapeptide precursor. The third retrocyclin geneencodes a variant nonapeptide with a glycine to arginine mutation.

In the Table 4, “Hs8_(—)19639” is the NCBI identifier for the Homosapiens chromosome 8 working draft sequence segment (Length=3,410,705bp). The retrocyclin nonapeptide is underlined.

TABLE 4 Chromosomal Hs8_19639 location Translated sequence Chromosome 82,814,641 (SEQ ID NO:125) V T P A Met R T F A L L T A Met L (SEQ IDNO:124) L L V A L Stop A Q A E P L Q A R A D E A A A Q E Q P G A D D Q EMet A H A F T W H E S A A L P L S 2,813,782 S D S A R G LR C I C G R G I C R L L Stop R R F G S C A F R G T L H R I C CChromosome 8 1,742,910 (SEQ ID NO:121) V T P A Met R T F A L L T A Met L(SEQ ID NO:120) L L V A L Stop A Q A E P L Q A R A D E A A A Q E Q P G AD D Q E Met A H A F T W H E S A A L P L S 1,742,051 S D S A R G LR C I C G R  R  I C R L L Stop R R F G S C A F R G T L H R I C CChromosome 8 1,478,367 (SEQ ID NO:123) V T P A Met R T F A L L T A Met L(SEQ ID NO:122) L L V A L Stop A Q A E H F R Q F L Met K L Q P R S S L EQ Met I H K W L Met L Y Met A Stop K C R S S A F 1,479,224 S D S A R G LR C I C G R G I C R L L Stop R R F G S C A F R G T L H R I C C

Based on the above information, the primate ancestor of Homo sapienscould have expressed three retrocyclins. The structures of thesepeptides, called Retrocyclin 1, 2 and 3 are shown in FIG. 9.

Three orangutan clones represent at least two different retrocyclingenes. The sequences are shown in FIG. 15. The stop codons in orangutanclone 19 are identical to those in human retrocyclin. Accordingly, clone19 also represents an expressed pseudogene. Overall, 132/143 (92.3%) oftranslated products (including stop codons) from orangutan clone 19 andthe human retrocyclin gene are identical. The translation products oforangutan clones 20 and 21 are identical in 141/143 (98.6%) sites. Bothclones lack a silencing stop codon in their signal sequence, and shouldbe capable of producing a functional demidefensin whose tandemnonapeptide elements (underlined) would produce a peptide identical tohuman retrocyclin. The predicted translation products of orangutan clone20 and human retrocyclin are identical in 129/143 (90.2%) of positions.All three orangutan clones, #19, 20 and 21 came from the DNA of a singleorangutan, It remains to be determined if the genes they represent arealleles, or if the retrocyclin locus has undergone duplication andadditional retrocyclin genes remain to be found.

As shown in FIG. 15, this portion of the human retrocyclin gene encodesfour stop codons (). The first of these occurs near the end of theputative signal sequence and should abort translation. The second stopcodon occurs after cysteine 3, and marks the end of the putativedemidefensin sequence. The third stop codon comes after the CCR residuesand marks the customary termination of an α-defensin. The final stopcodon occurs after the FES tripeptide in a non-expressed region of thegene.

One Gorilla retrocyclin clone has been sequenced. Its translationproduct is identical to human retrocyclin in 139/143 (97.2%). Thesequence is shown in an alignment with the human sequence in FIG. 15.The silencing stop codon () is present in the signal sequence.Consequently, this clone represents an expressed pseudogene.

Note that the chimp (Pan troglodytes) and the Bonobo (Pan paniscus)genes contain the first stop codon () in the signal sequence, but bothlack the retrocyclin-geherating stop codon after cysteine 3 in thedefensin-region. From these features, the chimp would appear to havesilenced an α-defensin gene. There is an additional mutation (cysteineto glycine) in the chimp's nonapeptide region (double underlined), whichwas presumably acquired after the gene had been silenced by the signalsequence mutation)

Unlike human retrocyclin, the pigtail and rhesus macaque genes lack astop codon () in their signal sequences. Both macaque genes haveacquired a stop codon in a nontranslated portion of their gene, betweencysteines 4 and 5 of the original defensin domain.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present invention is not entitled to antedate suchpublication by virtue of prior invention.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to those of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims.

                   #             SEQUENCE LISTING<160> NUMBER OF SEQ ID NOS: 125 <210> SEQ ID NO 1 <211> LENGTH: 18<212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 1Gly Ile Cys Arg Cys Ile Cys Gly Arg Gly Il #e Cys Arg Cys Ile Cys 1               5   #                10   #                15 Gly Arg<210> SEQ ID NO 2 <211> LENGTH: 18 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: synthetic variant <400> SEQUENCE: 2Gly Ile Cys Arg Cys Ile Cys Gly Lys Gly Il #e Cys Arg Cys Ile Cys 1               5   #                10   #                15 Gly Arg<210> SEQ ID NO 3 <211> LENGTH: 18 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: synthetic variant <400> SEQUENCE: 3Gly Ile Cys Arg Cys Tyr Cys Gly Arg Gly Il #e Cys Arg Cys Ile Cys 1               5   #                10   #                15 Gly Arg<210> SEQ ID NO 4 <211> LENGTH: 18 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: synthetic variant <400> SEQUENCE: 4Gly Ile Cys Arg Cys Ile Cys Gly Arg Gly Il #e Cys Arg Cys Tyr Cys 1               5   #                10   #                15 Gly Arg<210> SEQ ID NO 5 <211> LENGTH: 18 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: synthetic variant <400> SEQUENCE: 5Gly Tyr Cys Arg Cys Ile Cys Gly Arg Gly Il #e Cys Arg Cys Ile Cys 1               5   #                10   #                15 Gly Arg<210> SEQ ID NO 6 <211> LENGTH: 18 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: synthetic variant <400> SEQUENCE: 6Gly Ile Cys Arg Cys Ile Cys Gly Arg Gly Ty #r Cys Arg Cys Ile Cys 1               5   #                10   #                15 Gly Arg<210> SEQ ID NO 7 <211> LENGTH: 18 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: synthetic variant <400> SEQUENCE: 7Gly Ile Cys Tyr Cys Ile Cys Gly Arg Gly Il #e Cys Arg Cys Ile Cys 1               5   #                10   #                15 Gly Arg<210> SEQ ID NO 8 <211> LENGTH: 18 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: synthetic variant <400> SEQUENCE: 8Gly Ile Cys Ile Cys Ile Cys Gly Tyr Gly Il #e Cys Arg Cys Ile Cys 1               5   #                10   #                15 Gly Arg<210> SEQ ID NO 9 <211> LENGTH: 18 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: synthetic variant <400> SEQUENCE: 9Gly Ile Cys Ile Cys Ile Cys Gly Arg Gly Il #e Cys Tyr Cys Ile Cys 1               5   #                10   #                15 Gly Arg<210> SEQ ID NO 10 <211> LENGTH: 18 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: synthetic variant <400> SEQUENCE: 10Arg Gly Cys Ile Cys Arg Cys Ile Gly Arg Gl #y Cys Ile Cys Arg Cys 1               5   #                10   #                15 Ile Gly<210> SEQ ID NO 11 <211> LENGTH: 496 <212> TYPE: DNA<213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: CDS<222> LOCATION: (124)...(304) <223> OTHER INFORMATION: retrocyclin<400> SEQUENCE: 11ggagacccgg gacagaggac tgctgtctgc cctccctctt cactctgcct ac#cttgagga     60tctgtcaccc cagccatgag gaccttcgcc ctcctcactg ccatgcttct cc#tggtggcc    120 ctg tag gct cag gcg gag cca ctt cag gca ag#a gct gat gaa gct gca      168      *  Ala Gln Ala Glu Pro Leu G#ln Ala Arg Ala Asp Glu Ala Ala          1          #      5            #       10 gcc cag gag cag cct gga gca gat gat cag ga#a atg gct cat gcc ttt      216Ala Gln Glu Gln Pro Gly Ala Asp Asp Gln Gl #u Met Ala His Ala Phe 15                  # 20                  # 25                  # 30aca tgg cat gaa agt gcc gct ctt ccg ctt tc#a gac tca gcg aga ggc      264Thr Trp His Glu Ser Ala Ala Leu Pro Leu Se #r Asp Ser Ala Arg Gly                 35  #                 40  #                 45ttg agg tgc att tgc gga aga gga att tgc cg#t ttg tta t aacgtcgctt      314Leu Arg Cys Ile Cys Gly Arg Gly Ile Cys Ar #g Leu Leu             50      #             55tgggtcctgc gcctttcgtg gtacactcca ccggatctgc tgccgctgag ct#tgcagaat    374caagaaacat aagctcagaa tttactttga gagttaaaag aaattcttgt ta#ctcctgta    434ccttgtcctc catttccttt tctcatccaa aataaatacc ttgttgcaag at#ttctctct    494 tt                   #                  #                   #             496 <210> SEQ ID NO 12<211> LENGTH: 59 <212> TYPE: PRT <213> ORGANISM: Homo sapiens<400> SEQUENCE: 12 Ala Gln Ala Glu Pro Leu Gln Ala Arg Ala As#p Glu Ala Ala Ala Gln  1               5   #                10  #                15 Glu Gln Pro Gly Ala Asp Asp Gln Glu Met Al#a His Ala Phe Thr Trp             20       #            25      #            30 His Glu Ser Ala Ala Leu Pro Leu Ser Asp Se#r Ala Arg Gly Leu Arg         35           #        40          #        45 Cys Ile Cys Gly Arg Gly Ile Cys Arg Leu Le #u    50               #    55 <210> SEQ ID NO 13 <211> LENGTH: 97<212> TYPE: PRT <213> ORGANISM: Homo sapiens <220> FEATURE:<221> NAME/KEY: PEPTIDE <222> LOCATION: (1)...(97)<223> OTHER INFORMATION: Human defensin 4 <400> SEQUENCE: 13Met Arg Ile Ile Ala Leu Leu Ala Ala Ile Le #u Leu Val Ala Leu Gln 1               5   #                10   #                15Val Arg Ala Gly Pro Leu Gln Ala Arg Gly As #p Glu Ala Pro Gly Gln            20       #            25       #            30Glu Gln Arg Gly Pro Glu Asp Gln Asp Ile Se #r Ile Ser Phe Ala Trp        35           #        40           #        45Asp Lys Ser Ser Ala Leu Gln Val Ser Gly Se #r Thr Arg Gly Met Val    50               #    55               #    60Cys Ser Cys Arg Leu Val Phe Cys Arg Arg Th #r Glu Leu Arg Val Gly65                   #70                   #75                   #80Asn Cys Leu Ile Gly Gly Val Ser Phe Thr Ty #r Cys Cys Thr Arg Val                85   #                90   #                95 Asp<210> SEQ ID NO 14 <211> LENGTH: 500 <212> TYPE: DNA<213> ORGANISM: Macaca mulatta <220> FEATURE: <221> NAME/KEY: CDS<222> LOCATION: (95)...(325)<223> OTHER INFORMATION: theta defensin 1A precurs #or <220> FEATURE:<221> NAME/KEY: sig_peptide <222> LOCATION: (95)...(154) <220> FEATURE:<221> NAME/KEY: mat_peptide <222> LOCATION: (287)...(313)<223> OTHER INFORMATION: ligated to RTD1b in he#ad-to-tail orientation to       form the cyclic octadecapeptide RTD1#; RTD1 is       stabilized by three intramolecular d #isulfides<400> SEQUENCE: 14gacggctgct gttgctacag gagacccagg acagaggact gctgtctgca ct#ctctcttc     60 actctgccta acttgaggat ctgtcactcc agcc atg agg acc t#tc gcc ctc ctc    115                    #                  #  Met Arg Thr Phe Ala Leu Leu                    #                  #  -20                #  -15 acc gcc atg ctt ctc ctg gtg gcc ctg cac gc#t cag gca gag gca cgt      163Thr Ala Met Leu Leu Leu Val Ala Leu His Al #a Gln Ala Glu Ala Arg            -10       #            -5       #           1cag gca aga gct gat gaa gct gcc gcc cag ca#g cag cct gga aca gat      211Gln Ala Arg Ala Asp Glu Ala Ala Ala Gln Gl #n Gln Pro Gly Thr Asp     5              #      10             #      15gat cag gga atg gct cat tcc ttt aca tgg cc#t gaa aac gcc gct ctt      259Asp Gln Gly Met Ala His Ser Phe Thr Trp Pr #o Glu Asn Ala Ala Leu 20                  # 25                  # 30                  # 35cca ctt tca gag tca gcg aaa ggc ttg agg tg#c att tgc aca cga gga      307Pro Leu Ser Glu Ser Ala Lys Gly Leu Arg Cy #s Ile Cys Thr Arg Gly                 40  #                 45  #                 50ttc tgc cgt ttg tta taa tgtcaccttg ggtcctgcgc tt#ttcgtggt             355 Phe Cys Arg Leu Leu  *              55tgactccacc ggatctgctg ccgctgagct tccagaatca agaaaaatat gc#tcagaagt    415tactttgaga gttaaaagaa attcttgcta ctgctgtacc ttctcctcag tt#tccttttc    475 tcatcccaaa taaatacctt atcgc          #                   #              500 <210> SEQ ID NO 15<211> LENGTH: 76 <212> TYPE: PRT <213> ORGANISM: Macaca mulatta<220> FEATURE: <221> NAME/KEY: SIGNAL <222> LOCATION: (1)...(20)<400> SEQUENCE: 15 Met Arg Thr Phe Ala Leu Leu Thr Ala Met Le#u Leu Leu Val Ala Leu -20                 - #15                 -#10                 - #5 His Ala Gln Ala Glu Ala Arg Gln Ala Arg Al#a Asp Glu Ala Ala Ala                  1  #              5    #              10 Gln Gln Gln Pro Gly Thr Asp Asp Gln Gly Me#t Ala His Ser Phe Thr         15           #        20          #        25 Trp Pro Glu Asn Ala Ala Leu Pro Leu Ser Gl#u Ser Ala Lys Gly Leu     30               #    35              #    40 Arg Cys Ile Cys Thr Arg Gly Phe Cys Arg Le #u Leu45                   #50                   #55 <210> SEQ ID NO 16<211> LENGTH: 495 <212> TYPE: DNA <213> ORGANISM: Macaca mulatta<220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (90)...(320)<223> OTHER INFORMATION: theta defensin 1b precurs #or <220> FEATURE:<221> NAME/KEY: sig_peptide <222> LOCATION: (90)...(149) <220> FEATURE:<221> NAME/KEY: mat_peptide <222> LOCATION: (282)...(308)<223> OTHER INFORMATION: ligated to RTD1a in he#ad-to-tail orientation to       form the cyclic octadecapeptide RTD1#; RTD1 is       stabilized by three intramolecular d #isulfides<400> SEQUENCE: 16gaccgctgct cttgctacag gagacccggg acagaggact gctgtctgcc ct#ctctcttc     60 actctgccta acttgaggat ctgccagcc atg agg acc ttc gcc# ctc ctc acc      113                    #              Met Arg T#hr Phe Ala Leu Leu Thr                    #              -20   #              -15 gcc atg ctt ctc ctg gtg gcc ctg cac gct ca#g gca gag gca cgt cag      161Ala Met Leu Leu Leu Val Ala Leu His Ala Gl #n Ala Glu Ala Arg Gln        -10           #        -5           #       1gca aga gct gat gaa gct gcc gcc cag cag ca#g cct gga gca gat gat      209Ala Arg Ala Asp Glu Ala Ala Ala Gln Gln Gl #n Pro Gly Ala Asp Asp 5                  #  10                 #  15                 #  20cag gga atg gct cat tcc ttt aca cgg cct ga#a aac gcc gct ctt ccg      257Gln Gly Met Ala His Ser Phe Thr Arg Pro Gl #u Asn Ala Ala Leu Pro                 25  #                 30  #                 35ctt tca gag tca gcg aga ggc ttg agg tgc ct#t tgc aga cga gga gtt      305Leu Ser Glu Ser Ala Arg Gly Leu Arg Cys Le #u Cys Arg Arg Gly Val             40      #             45      #             50tgc caa ctg tta taa aggcgtttgg ggtcctgcgc ttttcgtgg#t tgactctgcc      360 Cys Gln Leu Leu  *          55ggatctgctg ccgctgagct tccagaatca agaaaaatac gctcagaagt ta#ctttgaga    420gttgaaagaa attcctgtta ctcctgtacc ttgtcctcaa tttccttttc tc#atcccaaa    480 taaatacctt ctcgc               #                  #                   #   495 <210> SEQ ID NO 17 <211> LENGTH: 76<212> TYPE: PRT <213> ORGANISM: Macaca mulatta <220> FEATURE:<221> NAME/KEY: SIGNAL <222> LOCATION: (1)...(20) <400> SEQUENCE: 17Met Arg Thr Phe Ala Leu Leu Thr Ala Met Le #u Leu Leu Val Ala Leu-20                 - #15                 - #10                 - #5His Ala Gln Ala Glu Ala Arg Gln Ala Arg Al #a Asp Glu Ala Ala Ala                 1  #              5     #              10Gln Gln Gln Pro Gly Ala Asp Asp Gln Gly Me #t Ala His Ser Phe Thr        15           #        20           #        25Arg Pro Glu Asn Ala Ala Leu Pro Leu Ser Gl #u Ser Ala Arg Gly Leu    30               #    35               #    40Arg Cys Leu Cys Arg Arg Gly Val Cys Gln Le #u Leu 45                  #50                   #55 <210> SEQ ID NO 18 <211> LENGTH: 54<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: synthetic sequence <400> SEQUENCE: 18aggtgcattt gcggaagagg aatttgcagg tgcatttgcg gaagaggaat tt#gc           54 <210> SEQ ID NO 19 <211> LENGTH: 9 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: generated by replacement  #of variants in                   #       consensus sequence <400> SEQUENCE: 19Arg Cys Ile Cys Gly Arg Gly Ile Cys  1               5<210> SEQ ID NO 20 <211> LENGTH: 9 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: generated by replacement  #of variants in                   #       consensus sequence <400> SEQUENCE: 20Arg Cys Leu Cys Gly Arg Gly Ile Cys  1               5<210> SEQ ID NO 21 <211> LENGTH: 9 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: generated by replacement  #of variants in                   #       consensus sequence <400> SEQUENCE: 21Arg Cys Ile Cys Arg Arg Gly Ile Cys  1               5<210> SEQ ID NO 22 <211> LENGTH: 9 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: generated by replacement  #of variants in                   #       consensus sequence <400> SEQUENCE: 22Arg Cys Ile Cys Thr Arg Gly Ile Cys  1               5<210> SEQ ID NO 23 <211> LENGTH: 9 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: generated by replacement  #of variants in                   #       consensus sequence <400> SEQUENCE: 23Arg Cys Ile Cys Val Arg Gly Ile Cys  1               5<210> SEQ ID NO 24 <211> LENGTH: 9 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: generated by replacement  #of variants in                   #       consensus sequence <400> SEQUENCE: 24Arg Cys Ile Cys Gly Leu Gly Ile Cys  1               5<210> SEQ ID NO 25 <211> LENGTH: 9 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: generated by replacement  #of variants in                   #       consensus sequence <400> SEQUENCE: 25Arg Cys Ile Cys Gly Arg Gly Val Cys  1               5<210> SEQ ID NO 26 <211> LENGTH: 9 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: generated by replacement  #of variants in                   #       consensus sequence <400> SEQUENCE: 26Arg Cys Ile Cys Gly Arg Gly Phe Cys  1               5<210> SEQ ID NO 27 <211> LENGTH: 9 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: generated by replacement  #of variants in                   #       consensus sequence <400> SEQUENCE: 27Arg Cys Leu Cys Arg Arg Gly Val Cys  1               5<210> SEQ ID NO 28 <211> LENGTH: 9 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: generated by replacement  #of variants in                   #       consensus sequence <400> SEQUENCE: 28Arg Cys Leu Cys Thr Arg Gly Ile Cys  1               5<210> SEQ ID NO 29 <211> LENGTH: 9 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: generated by replacement  #of variants in                   #       consensus sequence <400> SEQUENCE: 29Arg Cys Leu Cys Val Arg Gly Ile Cys  1               5<210> SEQ ID NO 30 <211> LENGTH: 9 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: generated by replacement  #of variants in                   #       consensus sequence <400> SEQUENCE: 30Arg Cys Leu Cys Gly Leu Gly Val Cys  1               5<210> SEQ ID NO 31 <211> LENGTH: 9 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: generated by replacement  #of variants in                   #       consensus sequence <400> SEQUENCE: 31Arg Cys Leu Cys Gly Arg Gly Val Cys  1               5<210> SEQ ID NO 32 <211> LENGTH: 9 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: generated by replacement  #of variants in                   #       consensus sequence <400> SEQUENCE: 32Arg Cys Leu Cys Gly Arg Gly Phe Cys  1               5<210> SEQ ID NO 33 <211> LENGTH: 9 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: generated by replacement  #of variants in                   #       consensus sequence <400> SEQUENCE: 33Arg Cys Ile Cys Arg Arg Gly Val Cys  1               5<210> SEQ ID NO 34 <211> LENGTH: 9 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: generated by replacement  #of variants in                   #       consensus sequence <400> SEQUENCE: 34Arg Cys Ile Cys Arg Arg Gly Phe Cys  1               5<210> SEQ ID NO 35 <211> LENGTH: 9 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: generated by replacement  #of variants in                   #       consensus sequence <400> SEQUENCE: 35Arg Cys Ile Cys Thr Arg Gly Val Cys  1               5<210> SEQ ID NO 36 <211> LENGTH: 9 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: generated by replacement  #of variants in                   #       consensus sequence <400> SEQUENCE: 36Arg Cys Ile Cys Thr Arg Gly Phe Cys  1               5<210> SEQ ID NO 37 <211> LENGTH: 9 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: generated by replacement  #of variants in                   #       consensus sequence <400> SEQUENCE: 37Arg Cys Ile Cys Thr Leu Gly Ile Cys  1               5<210> SEQ ID NO 38 <211> LENGTH: 9 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: generated by replacement  #of variants in                   #       consensus sequence <400> SEQUENCE: 38Arg Cys Ile Cys Val Leu Gly Phe Cys  1               5<210> SEQ ID NO 39 <211> LENGTH: 9 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: generated by replacement  #of variants in                   #       consensus sequence <400> SEQUENCE: 39Arg Cys Ile Cys Arg Leu Gly Ile Cys  1               5<210> SEQ ID NO 40 <211> LENGTH: 9 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: generated by replacement  #of variants in                   #       consensus sequence <400> SEQUENCE: 40Arg Cys Ile Cys Val Arg Gly Val Cys  1               5<210> SEQ ID NO 41 <211> LENGTH: 9 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: generated by replacement  #of variants in                   #       consensus sequence <400> SEQUENCE: 41Arg Cys Ile Cys Gly Arg Gly Phe Cys  1               5<210> SEQ ID NO 42 <211> LENGTH: 9 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: generated by replacement  #of variants in                   #       consensus sequence <400> SEQUENCE: 42Arg Cys Ile Cys Gly Leu Gly Phe Cys  1               5<210> SEQ ID NO 43 <211> LENGTH: 9 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: generated by replacement  #of variants in                   #       consensus sequence <400> SEQUENCE: 43Arg Cys Ile Cys Gly Leu Gly Val Cys  1               5<210> SEQ ID NO 44 <211> LENGTH: 9 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: generated by replacement  #of variants in                   #       consensus sequence <400> SEQUENCE: 44Arg Cys Leu Cys Arg Leu Gly Ile Cys  1               5<210> SEQ ID NO 45 <211> LENGTH: 9 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: generated by replacement  #of variants in                   #       consensus sequence <400> SEQUENCE: 45Arg Cys Leu Cys Arg Arg Gly Val Cys  1               5<210> SEQ ID NO 46 <211> LENGTH: 9 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: generated by replacement  #of variants in                   #       consensus sequence <400> SEQUENCE: 46Arg Cys Leu Cys Arg Arg Gly Phe Cys  1               5<210> SEQ ID NO 47 <211> LENGTH: 9 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: generated by replacement  #of variants in                   #       consensus sequence <400> SEQUENCE: 47Arg Cys Leu Cys Thr Leu Gly Ile Cys  1               5<210> SEQ ID NO 48 <211> LENGTH: 9 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: generated by replacement  #of variants in                   #       consensus sequence <400> SEQUENCE: 48Arg Cys Leu Cys Thr Arg Gly Val Cys  1               5<210> SEQ ID NO 49 <211> LENGTH: 9 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: generated by replacement  #of variants in                   #       consensus sequence <400> SEQUENCE: 49Arg Cys Leu Cys Thr Arg Gly Phe Cys  1               5<210> SEQ ID NO 50 <211> LENGTH: 9 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: generated by replacement  #of variants in                   #       consensus sequence <400> SEQUENCE: 50Arg Cys Leu Cys Val Leu Gly Ile Cys  1               5<210> SEQ ID NO 51 <211> LENGTH: 9 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: generated by replacement  #of variants in                   #       consensus sequence <400> SEQUENCE: 51Arg Cys Leu Cys Val Arg Gly Val Cys  1               5<210> SEQ ID NO 52 <211> LENGTH: 9 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: generated by replacement  #of variants in                   #       consensus sequence <400> SEQUENCE: 52Arg Cys Ile Cys Gly Arg Gly Ile Cys  1               5<210> SEQ ID NO 53 <211> LENGTH: 9 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: generated by replacement  #of variants in                   #       consensus sequence <400> SEQUENCE: 53Arg Cys Ile Cys Arg Leu Gly Val Cys  1               5<210> SEQ ID NO 54 <211> LENGTH: 9 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: generated by replacement  #of variants in                   #       consensus sequence <400> SEQUENCE: 54Arg Cys Ile Cys Arg Leu Gly Phe Cys  1               5<210> SEQ ID NO 55 <211> LENGTH: 9 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: generated by replacement  #of variants in                   #       consensus sequence <400> SEQUENCE: 55Arg Cys Ile Cys Thr Leu Gly Val Cys  1               5<210> SEQ ID NO 56 <211> LENGTH: 9 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: generated by replacement  #of variants in                   #       consensus sequence <400> SEQUENCE: 56Arg Cys Ile Cys Thr Leu Gly Phe Cys  1               5<210> SEQ ID NO 57 <211> LENGTH: 9 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: generated by replacement  #of variants in                   #       consensus sequence <400> SEQUENCE: 57Arg Cys Ile Cys Val Leu Gly Val Cys  1               5<210> SEQ ID NO 58 <211> LENGTH: 9 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: generated by replacement  #of variants in                   #       consensus sequence <400> SEQUENCE: 58Arg Cys Ile Cys Val Leu Gly Phe Cys  1               5<210> SEQ ID NO 59 <211> LENGTH: 9 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: generated by replacement  #of variants in                   #       consensus sequence <400> SEQUENCE: 59Arg Cys Leu Cys Gly Leu Gly Val Cys  1               5<210> SEQ ID NO 60 <211> LENGTH: 9 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: generated by replacement  #of variants in                   #       consensus sequence <400> SEQUENCE: 60Arg Cys Leu Cys Gly Leu Gly Ile Cys  1               5<210> SEQ ID NO 61 <211> LENGTH: 9 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: generated by replacement  #of variants in                   #       consensus sequence <400> SEQUENCE: 61Arg Cys Leu Cys Thr Leu Gly Val Cys  1               5<210> SEQ ID NO 62 <211> LENGTH: 9 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: generated by replacement  #of variants in                   #       consensus sequence <400> SEQUENCE: 62Arg Cys Leu Cys Thr Leu Gly Ile Cys  1               5<210> SEQ ID NO 63 <211> LENGTH: 9 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: generated by replacement  #of variants in                   #       consensus sequence <400> SEQUENCE: 63Arg Cys Leu Cys Val Leu Gly Val Cys  1               5<210> SEQ ID NO 64 <211> LENGTH: 9 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: generated by replacement  #of variants in                   #       consensus sequence <400> SEQUENCE: 64Arg Cys Leu Cys Val Leu Gly Ile Cys  1               5<210> SEQ ID NO 65 <211> LENGTH: 140 <212> TYPE: PRT<213> ORGANISM: Homo sapiens <400> SEQUENCE: 65Val Thr Pro Ala Met Arg Thr Phe Ala Leu Le #u Thr Ala Met Leu Leu 1               5   #                10   #                15Leu Val Ala Leu Ala Gln Ala Glu Pro Leu Gl #n Ala Arg Ala Asp Glu            20       #            25       #            30Ala Ala Ala Gln Glu Gln Pro Gly Ala Asp As #p Gln Glu Met Ala His        35           #        40           #        45Ala Phe Thr Trp His Glu Ser Ala Ala Leu Pr #o Leu Ser Asp Ser Ala    50               #    55               #    60Arg Gly Leu Arg Cys Ile Cys Gly Arg Gly Il #e Cys Arg Leu Leu Arg65                   #70                   #75                   #80Arg Phe Gly Ser Cys Ala Phe Arg Gly Thr Le #u His Arg Ile Cys Cys                85   #                90   #                95Arg Ala Cys Arg Ile Lys Lys His Lys Leu Ar #g Ile Tyr Phe Glu Ser            100       #           105       #           110Lys Lys Phe Leu Leu Leu Leu Tyr Leu Val Le #u His Phe Leu Phe Ser        115           #       120           #       125Ser Lys Ile Asn Thr Leu Leu Gln Asp Phe Se #r Leu     130              #   135               #   140 <210> SEQ ID NO 66 <211> LENGTH: 140<212> TYPE: PRT <213> ORGANISM: Orangutan <400> SEQUENCE: 66Val Thr Pro Ala Met Arg Thr Phe Ala Leu Le #u Ala Ala Met Leu Leu 1               5   #                10   #                15Leu Val Ala Leu Ala Glu Ala Glu Pro Leu Gl #n Ala Arg Ala Asp Glu            20       #            25       #            30Thr Ala Ala Gln Glu Gln Pro Gly Ala Asp As #p Gln Glu Met Ala His        35           #        40           #        45Ala Phe Thr Trp Asp Glu Ser Ala Thr Leu Pr #o Leu Ser Asp Ser Ala    50               #    55               #    60Arg Gly Leu Arg Cys Ile Cys Arg Arg Gly Va #l Cys Arg Phe Leu Arg65                   #70                   #75                   #80His Leu Gly Ser Cys Ala Phe Arg Gly Thr Le #u His Arg Ile Cys Cys                85   #                90   #                95Arg Ala Cys Arg Ile Lys Lys Asn Lys Leu Ar #g Ile Tyr Phe Glu Ser            100       #           105       #           110Lys Lys Phe Val Phe Leu Leu Tyr Leu Ala Le #u His Phe Leu Phe Ser        115           #       120           #       125Ser Lys Ile Asn Thr Leu Leu Gln Asp Phe Cy #s Leu     130              #   135               #   140 <210> SEQ ID NO 67 <211> LENGTH: 141<212> TYPE: PRT <213> ORGANISM: Orangutan <400> SEQUENCE: 67Val Thr Pro Ala Met Arg Thr Phe Thr Val Le #u Ala Ala Met Leu Leu 1               5   #                10   #                15Val Val Ala Leu Gln Ala Gln Ala Glu Pro Le #u Arg Ala Arg Ala Asp            20       #            25       #            30Glu Thr Ala Ala Gln Glu Gln Pro Gly Ala As #p Asp Gln Glu Met Ala        35           #        40           #        45His Ala Phe Thr Trp Asp Glu Ser Ala Ala Le #u Pro Leu Ser Asp Ser    50               #    55               #    60Ala Arg Gly Leu Arg Cys Ile Cys Arg Arg Gl #y Val Cys Arg Phe Leu65                   #70                   #75                   #80Arg His Leu Gly Ser Cys Ala Phe Arg Gly Th #r Leu His Arg Ile Cys                85   #                90   #                95Cys Arg Ala Cys Arg Ile Lys Lys Asn Lys Le #u Arg Ile Tyr Phe Glu            100       #           105       #           110Ser Lys Lys Phe Val Phe Leu Leu Tyr Leu Al #a Leu His Phe Leu Phe        115           #       120           #       125Ser Ser Lys Ile Asn Thr Leu Leu Gln Asp Ph #e Cys Leu    130               #   135               #   140 <210> SEQ ID NO 68<211> LENGTH: 141 <212> TYPE: PRT <213> ORGANISM: Orangutan<400> SEQUENCE: 68 Val Thr Pro Ala Met Arg Thr Phe Thr Val Le#u Ala Ala Met Leu Leu  1               5   #                10  #                15 Val Val Ala Leu Gln Ala Gln Ala Glu Pro Le#u Arg Ala Arg Ala Asp             20       #            25      #            30 Glu Thr Ala Ala Gln Glu Gln Pro Gly Ala As#p Asp Gln Glu Met Ala         35           #        40          #        45 His Ala Phe Thr Trp Asp Glu Ser Ala Ala Le#u Pro Leu Ser Asp Ser     50               #    55              #    60 Ala Arg Gly Leu Arg Cys Ile Cys Arg Arg Gl#y Val Cys Arg Leu Leu 65                   #70                  #75                   #80 Arg His Phe Gly Ser Cys Ala Phe Arg Gly Th#r Leu His Arg Ile Cys                 85   #                90  #                95 Cys Arg Ala Cys Arg Ile Lys Lys Asn Lys Le#u Arg Ile Tyr Phe Glu             100       #           105      #           110 Ser Lys Lys Phe Leu Phe Leu Leu Tyr Leu Al#a Leu His Phe Leu Phe         115           #       120          #       125 Ser Ser Lys Ile Asn Thr Leu Leu Gln Asp Ph #e Cys Leu    130               #   135               #   140 <210> SEQ ID NO 69<211> LENGTH: 140 <212> TYPE: PRT <213> ORGANISM: Gorilla<400> SEQUENCE: 69 Val Thr Pro Ala Met Arg Thr Phe Ala Leu Le#u Thr Ala Met Leu Leu  1               5   #                10  #                15 Leu Val Asp Leu Ala Gln Ala Glu Pro Leu Gl#n Ala Arg Ala Asp Glu             20       #            25      #            30 Ala Ala Ala Gln Glu Gln Pro Gly Ala Asp As#p Gln Glu Met Ala His         35           #        40          #        45 Ala Phe Thr Trp Asp Glu Ser Ala Ala Leu Pr#o Leu Ser Asp Ser Ala     50               #    55              #    60 Arg Gly Leu Arg Cys Ile Cys Gly Arg Gly Il#e Cys Arg Leu Leu Arg 65                   #70                  #75                   #80 Arg Phe Gly Ser Cys Ala Phe Arg Gly Thr Le#u His Arg Ile Cys Cys                 85   #                90  #                95 Arg Ala Cys Arg Ile Lys Lys Asn Lys Leu Ar#g Ile Tyr Phe Glu Thr             100       #           105      #           110 Lys Lys Phe Leu Leu Leu Leu Tyr Leu Val Le#u His Phe Leu Phe Ser         115           #       120          #       125 Ser Lys Ile Asn Thr Leu Leu Gln Asp Phe Cy #s Leu    130               #   135               #   140 <210> SEQ ID NO 70<211> LENGTH: 141 <212> TYPE: PRT <213> ORGANISM: Champanzee<400> SEQUENCE: 70 Val Thr Pro Ala Met Arg Thr Phe Ala Leu Le#u Thr Ala Met Leu Leu  1               5   #                10  #                15 Leu Val Ala Leu Ala Gln Ala Glu Pro Leu Gl#n Ala Arg Ala Asp Glu             20       #            25      #            30 Ala Ala Ala Gln Glu Gln Pro Gly Ala Asp As#p Gln Glu Met Ala His         35           #        40          #        45 Ala Phe Thr Trp Asp Glu Ser Ala Ala Leu Pr#o Leu Ser Asp Ser Ala     50               #    55              #    60 Arg Gly Leu Arg Cys Ile Gly Gly Arg Gly Il#e Cys Gly Leu Leu Gln 65                   #70                  #75                   #80 Arg Arg Phe Gly Ser Cys Ala Phe Arg Gly Th#r Leu His Arg Ile Cys                 85   #                90  #                95 Cys Arg Ala Cys Arg Ile Lys Lys Asn Lys Le#u Arg Ile Tyr Ser Glu             100       #           105      #           110 Ser Lys Lys Phe Leu Leu Leu Leu Tyr Leu Va#l Leu His Phe Leu Phe         115           #       120          #       125 Ser Ser Lys Ile Asn Thr Leu Leu Gln Asp Ph #e Ser Leu    130               #   135               #   140 <210> SEQ ID NO 71<211> LENGTH: 141 <212> TYPE: PRT <213> ORGANISM: Chimpanzee<400> SEQUENCE: 71 Val Thr Pro Ala Met Arg Thr Phe Ala Leu Le#u Thr Ala Met Leu Leu  1               5   #                10  #                15 Leu Val Ala Leu Ala Gln Ala Glu Pro Leu Gl#n Ala Arg Ala Asp Glu             20       #            25      #            30 Ala Ala Ala Gln Glu Gln Pro Gly Ala Asp As#p Gln Glu Met Ala His         35           #        40          #        45 Ala Phe Thr Trp Asp Glu Ser Ala Ala Leu Pr#o Leu Ser Asp Ser Ala     50               #    55              #    60 Arg Gly Leu Arg Cys Ile Gly Gly Arg Gly Il#e Cys Gly Leu Leu Gln 65                   #70                  #75                   #80 Arg Arg Val Gly Ser Cys Ala Phe Arg Gly Th#r Leu His Arg Ile Cys                 85   #                90  #                95 Cys Arg Ala Cys Arg Ile Lys Lys Asn Lys Le#u Arg Ile Tyr Ser Glu             100       #           105      #           110 Ser Lys Lys Phe Leu Leu Leu Leu Tyr Leu Va#l Leu His Phe Leu Phe         115           #       120          #       125 Ser Ser Lys Ile Asn Thr Ser Leu Gln Asp Ph #e Ser Leu    130               #   135               #   140 <210> SEQ ID NO 72<211> LENGTH: 141 <212> TYPE: PRT <213> ORGANISM: Rhesus monkey<220> FEATURE: <221> NAME/KEY: VARIANT <222> LOCATION: 113<223> OTHER INFORMATION: Xaa = Any Amino Aci #d <400> SEQUENCE: 72Val Thr Pro Ala Met Arg Thr Phe Ala Leu Le #u Thr Ala Met Leu Leu 1               5   #                10   #                15Leu Val Ala Leu His Ala Gln Ala Glu Ala Ar #g Gln Ala Arg Ala Asp            20       #            25       #            30Glu Ala Ala Ala Gln Gln Gln Pro Gly Ala As #p Asp Gln Gly Met Ala        35           #        40           #        45His Ser Phe Thr Arg Pro Glu Asn Ala Ala Le #u Pro Leu Ser Glu Ser    50               #    55               #    60Ala Arg Gly Leu Arg Cys Leu Cys Arg Arg Gl #y Val Cys Gln Leu Leu65                   #70                   #75                   #80Arg Arg Leu Gly Ser Cys Ala Phe Arg Gly Le #u Cys Arg Ile Cys Cys                85   #                90   #                95Arg Ala Ser Arg Ile Lys Lys Asn Thr Leu Ar #g Ser Tyr Phe Glu Ser            100       #           105       #           110Xaa Lys Lys Phe Leu Leu Leu Leu Tyr Leu Va #l Leu Asn Phe Leu Phe        115           #       120           #       125Ser Ser Gln Ile Asn Thr Phe Ser Gln Asp Ph #e Cys Leu    130               #   135               #   140 <210> SEQ ID NO 73<211> LENGTH: 141 <212> TYPE: PRT <213> ORGANISM: Pig-tailed macaque<220> FEATURE: <221> NAME/KEY: VARIANT <222> LOCATION: 113<223> OTHER INFORMATION: Xaa = Any Amino Aci #d <400> SEQUENCE: 73Val Thr Pro Ala Met Arg Thr Phe Ala Leu Le #u Thr Ala Met Leu Leu 1               5   #                10   #                15Leu Val Ala Leu His Ala Gln Ala Glu Ala Ar #g Gln Ala Arg Ala Asp            20       #            25       #            30Glu Ala Ala Ala Gln Gln Gln Pro Gly Ala As #p Asp Gln Gly Met Ala        35           #        40           #        45His Ser Phe Thr Arg Pro Glu Asn Ala Ala Le #u Pro Leu Ser Glu Ser    50               #    55               #    60Ala Arg Gly Leu Arg Cys Ile Cys Arg Arg Gl #y Val Cys Gln Leu Leu65                   #70                   #75                   #80Arg Arg Leu Gly Ser Cys Ala Phe Arg Gly Le #u Cys Arg Ile Cys Cys                85   #                90   #                95Arg Ala Ser Arg Ile Lys Lys Asn Thr Leu Ar #g Ser Tyr Phe Glu Ser            100       #           105       #           110Xaa Lys Lys Phe Leu Leu Leu Leu Tyr Leu Va #l Leu Asn Phe Leu Phe        115           #       120           #       125Ser Ser Gln Ile Asn Thr Phe Ser Gln Asp Ph #e Cys Leu    130               #   135               #   140 <210> SEQ ID NO 74<211> LENGTH: 9 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: expansion of variant resi #dues.<400> SEQUENCE: 74 Arg Cys Ile Cys Gly Arg Arg Ile Cys 1               5 <210> SEQ ID NO 75 <211> LENGTH: 9 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: expansion of variant resi #dues.<400> SEQUENCE: 75 Arg Cys Leu Cys Gly Arg Arg Ile Cys 1               5 <210> SEQ ID NO 76 <211> LENGTH: 9 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: expansion of variant resi #dues.<400> SEQUENCE: 76 Arg Cys Ile Cys Arg Arg Arg Ile Cys 1               5 <210> SEQ ID NO 77 <211> LENGTH: 9 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: expansion of variant resi #dues.<400> SEQUENCE: 77 Arg Cys Ile Cys Thr Arg Arg Ile Cys 1               5 <210> SEQ ID NO 78 <211> LENGTH: 9 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: expansion of variant resi #dues.<400> SEQUENCE: 78 Arg Cys Ile Cys Val Arg Arg Ile Cys 1               5 <210> SEQ ID NO 79 <211> LENGTH: 9 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: expansion of variant resi #dues.<400> SEQUENCE: 79 Arg Cys Ile Cys Gly Leu Arg Ile Cys 1               5 <210> SEQ ID NO 80 <211> LENGTH: 9 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: expansion of variant resi #dues.<400> SEQUENCE: 80 Arg Cys Ile Cys Gly Arg Arg Val Cys 1               5 <210> SEQ ID NO 81 <211> LENGTH: 9 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: expansion of variant resi #dues.<400> SEQUENCE: 81 Arg Cys Ile Cys Gly Arg Arg Phe Cys 1               5 <210> SEQ ID NO 82 <211> LENGTH: 9 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: expansion of variant resi #dues.<400> SEQUENCE: 82 Arg Cys Leu Cys Arg Arg Arg Val Cys 1               5 <210> SEQ ID NO 83 <211> LENGTH: 9 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: expansion of variant resi #dues.<400> SEQUENCE: 83 Arg Cys Leu Cys Thr Arg Arg Ile Cys 1               5 <210> SEQ ID NO 84 <211> LENGTH: 9 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: expansion of variant resi #dues.<400> SEQUENCE: 84 Arg Cys Leu Cys Val Arg Arg Ile Cys 1               5 <210> SEQ ID NO 85 <211> LENGTH: 9 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: expansion of variant resi #dues.<400> SEQUENCE: 85 Arg Cys Leu Cys Gly Leu Arg Val Cys 1               5 <210> SEQ ID NO 86 <211> LENGTH: 9 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: expansion of variant resi #dues.<400> SEQUENCE: 86 Arg Cys Leu Cys Gly Arg Arg Val Cys 1               5 <210> SEQ ID NO 87 <211> LENGTH: 9 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: expansion of variant resi #dues.<400> SEQUENCE: 87 Arg Cys Leu Cys Gly Arg Arg Phe Cys 1               5 <210> SEQ ID NO 88 <211> LENGTH: 9 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: expansion of variant resi #dues.<400> SEQUENCE: 88 Arg Cys Ile Cys Arg Arg Arg Val Cys 1               5 <210> SEQ ID NO 89 <211> LENGTH: 9 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: expansion of variant resi #dues.<400> SEQUENCE: 89 Arg Cys Ile Cys Arg Arg Arg Phe Cys 1               5 <210> SEQ ID NO 90 <211> LENGTH: 9 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: expansion of variant resi #dues.<400> SEQUENCE: 90 Arg Cys Ile Cys Thr Arg Arg Val Cys 1               5 <210> SEQ ID NO 91 <211> LENGTH: 9 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: expansion of variant resi #dues.<400> SEQUENCE: 91 Arg Cys Ile Cys Thr Arg Arg Phe Cys 1               5 <210> SEQ ID NO 92 <211> LENGTH: 9 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: expansion of variant resi #dues.<400> SEQUENCE: 92 Arg Cys Ile Cys Thr Leu Arg Ile Cys 1               5 <210> SEQ ID NO 93 <211> LENGTH: 9 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: expansion of variant resi #dues.<400> SEQUENCE: 93 Arg Cys Ile Cys Val Leu Arg Phe Cys 1               5 <210> SEQ ID NO 94 <211> LENGTH: 9 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: expansion of variant resi #dues.<400> SEQUENCE: 94 Arg Cys Ile Cys Arg Leu Arg Ile Cys 1               5 <210> SEQ ID NO 95 <211> LENGTH: 9 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: expansion of variant resi #dues.<400> SEQUENCE: 95 Arg Cys Ile Cys Val Arg Arg Val Cys 1               5 <210> SEQ ID NO 96 <211> LENGTH: 9 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: expansion of variant resi #dues.<400> SEQUENCE: 96 Arg Cys Ile Cys Gly Arg Arg Phe Cys 1               5 <210> SEQ ID NO 97 <211> LENGTH: 9 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: expansion of variant resi #dues.<400> SEQUENCE: 97 Arg Cys Ile Cys Gly Leu Arg Phe Cys 1               5 <210> SEQ ID NO 98 <211> LENGTH: 9 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: expansion of variant resi #dues.<400> SEQUENCE: 98 Arg Cys Ile Cys Gly Leu Arg Val Cys 1               5 <210> SEQ ID NO 99 <211> LENGTH: 9 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: expansion of variant resi #dues.<400> SEQUENCE: 99 Arg Cys Leu Cys Arg Leu Arg Ile Cys 1               5 <210> SEQ ID NO 100 <211> LENGTH: 9 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: expansion of variant resi #dues.<400> SEQUENCE: 100 Arg Cys Leu Cys Arg Arg Arg Val Cys 1               5 <210> SEQ ID NO 101 <211> LENGTH: 9 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: expansion of variant resi #dues.<400> SEQUENCE: 101 Arg Cys Leu Cys Arg Arg Arg Phe Cys 1               5 <210> SEQ ID NO 102 <211> LENGTH: 9 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: expansion of variant resi #dues.<400> SEQUENCE: 102 Arg Cys Leu Cys Thr Leu Arg Ile Cys 1               5 <210> SEQ ID NO 103 <211> LENGTH: 9 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: expansion of variant resi #dues.<400> SEQUENCE: 103 Arg Cys Leu Cys Gly Arg Arg Val Cys 1               5 <210> SEQ ID NO 104 <211> LENGTH: 9 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: expansion of variant resi #dues.<400> SEQUENCE: 104 Arg Cys Leu Cys Thr Arg Arg Phe Cys 1               5 <210> SEQ ID NO 105 <211> LENGTH: 9 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: expansion of variant resi #dues.<400> SEQUENCE: 105 Arg Cys Leu Cys Val Leu Arg Ile Cys 1               5 <210> SEQ ID NO 106 <211> LENGTH: 9 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: expansion of variant resi #dues.<400> SEQUENCE: 106 Arg Cys Leu Cys Val Arg Arg Val Cys 1               5 <210> SEQ ID NO 107 <211> LENGTH: 9 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: expansion of variant resi #dues.<400> SEQUENCE: 107 Arg Cys Ile Cys Gly Arg Arg Ile Cys 1               5 <210> SEQ ID NO 108 <211> LENGTH: 9 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: expansion of variant resi #dues.<400> SEQUENCE: 108 Arg Cys Ile Cys Arg Leu Arg Val Cys 1               5 <210> SEQ ID NO 109 <211> LENGTH: 9 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: expansion of variant resi #dues.<400> SEQUENCE: 109 Arg Cys Ile Cys Arg Leu Arg Phe Cys 1               5 <210> SEQ ID NO 110 <211> LENGTH: 9 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: expansion of variant resi #dues.<400> SEQUENCE: 110 Arg Cys Ile Cys Thr Leu Arg Val Cys 1               5 <210> SEQ ID NO 111 <211> LENGTH: 9 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: expansion of variant resi #dues.<400> SEQUENCE: 111 Arg Cys Ile Cys Thr Leu Arg Phe Cys 1               5 <210> SEQ ID NO 112 <211> LENGTH: 9 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: expansion of variant resi #dues.<400> SEQUENCE: 112 Arg Cys Ile Cys Val Leu Arg Val Cys 1               5 <210> SEQ ID NO 113 <211> LENGTH: 9 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: expansion of variant resi #dues.<400> SEQUENCE: 113 Arg Cys Ile Cys Val Leu Arg Phe Cys 1               5 <210> SEQ ID NO 114 <211> LENGTH: 9 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: expansion of variant resi #dues.<400> SEQUENCE: 114 Arg Cys Leu Cys Gly Leu Arg Val Cys 1               5 <210> SEQ ID NO 115 <211> LENGTH: 9 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: expansion of variant resi #dues.<400> SEQUENCE: 115 Arg Cys Leu Cys Gly Leu Arg Ile Cys 1               5 <210> SEQ ID NO 116 <211> LENGTH: 9 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: expansion of variant resi #dues.<400> SEQUENCE: 116 Arg Cys Leu Cys Thr Leu Arg Val Cys 1               5 <210> SEQ ID NO 117 <211> LENGTH: 9 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: expansion of variant resi #dues.<400> SEQUENCE: 117 Arg Cys Leu Cys Thr Leu Arg Ile Cys 1               5 <210> SEQ ID NO 118 <211> LENGTH: 9 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: expansion of variant resi #dues.<400> SEQUENCE: 118 Arg Cys Leu Cys Val Leu Arg Val Cys 1               5 <210> SEQ ID NO 119 <211> LENGTH: 9 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: expansion of variant resi #dues.<400> SEQUENCE: 119 Arg Cys Leu Cys Val Leu Arg Ile Cys 1               5 <210> SEQ ID NO 120 <211> LENGTH: 437 <212> TYPE: DNA<213> ORGANISM: Homo sapiens <400> SEQUENCE: 120gtcaccccag ccatgaggac cttcgccctc ctcactgcca tgcttctcct gg#tggccctg     60taggctcagg cggagccact tcaggcaaga gctgatgaag ctgcagccca gg#agcagcct    120ggagcagatg atcaggaaat ggctcatgcc tttacatggc atgaaagtgc cg#ctcttccg    180ttttcagtca gactcagcga gaggcttgag gtgcatttgc ggaagaggaa tt#tgccgttt    240gttataacgt cgctttgggt cctgcgcctt tcgtggtaca ctccaccggg tc#tgctgccg    300ctgaacttgc agaatcaaga aaaataagct cagaatttac tttgagagtt aa#aagaaatt    360cttgttactc ctgtaccttg tcctccattt ccttttctca tccaaaataa at#accttgtt    420 gcaagatttc tctcttt              #                  #                   #  437 <210> SEQ ID NO 121 <211> LENGTH: 118<212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 121Val Thr Pro Ala Met Arg Thr Phe Ala Leu Le #u Thr Ala Met Leu Leu 1               5   #                10   #                15Leu Val Ala Leu Ala Gln Ala Glu Pro Leu Gl #n Ala Arg Ala Asp Glu            20       #            25       #            30Ala Ala Ala Gln Glu Gln Ser Asp Ser Ala Ar #g Gly Leu Arg Cys Ile        35           #        40           #        45Cys Gly Arg Gly Ile Cys Arg Leu Leu Arg Ar #g Phe Gly Ser Cys Ala    50               #    55               #    60Phe Arg Gly Thr Leu His Arg Val Cys Cys Ar #g Thr Cys Arg Ile Lys65                   #70                   #75                   #80Lys Asn Lys Leu Arg Ile Tyr Phe Glu Ser Ly #s Lys Phe Leu Leu Leu                85   #                90   #                95Leu Tyr Leu Val Leu His Phe Leu Phe Ser Se #r Lys Ile Asn Thr Leu            100       #           105       #           110Leu Gln Asp Phe Ser Leu         115 <210> SEQ ID NO 122<211> LENGTH: 437 <212> TYPE: DNA <213> ORGANISM: Homo sapiens<400> SEQUENCE: 122gtcaccccag ccatgaggac ctttgccctc ctcactgcca tgcttctcct gg#tggccctg     60taggctcagg cagagccact tcaggcaaga gctgatgaag ctgcagccca gg#agcagcct    120ggagcagatg atcaggaaat ggctcatgcc tttacatggc atgaaagtgc cg#ctcttccg    180ctttcagtca gactcagcga gaggcttgag gtgcatttgc ggaagaagaa tt#tgccgttt    240gttataacgt cgctttgggt cctgcgcctt tcgtggtaca ctccaccgga tc#tgctgccg    300ctgagcttgc agaatcaaga aacataagct cagaatttac tttgagagtt aa#aagaaatt    360cttgttactc ctgtaccttg tcctccattt ccttttctca tccaaaataa at#accttgtt    420 gcaagatttc tctcttt              #                  #                   #  437 <210> SEQ ID NO 123 <211> LENGTH: 141<212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 123Val Thr Pro Ala Met Arg Thr Phe Ala Leu Le #u Thr Ala Met Leu Leu 1               5   #                10   #                15Leu Val Ala Leu Ala Gln Ala Glu Pro Leu Gl #n Ala Arg Ala Asp Glu            20       #            25       #            30Ala Ala Ala Gln Glu Gln Pro Gly Ala Asp As #p Gln Glu Met Ala His        35           #        40           #        45Ala Phe Thr Trp His Glu Ser Ala Ala Leu Pr #o Leu Ser Ser Asp Ser    50               #    55               #    60Ala Arg Gly Leu Arg Cys Ile Cys Gly Arg Ar #g Ile Cys Arg Leu Leu65                   #70                   #75                   #80Arg Arg Phe Gly Ser Cys Ala Phe Arg Gly Th #r Leu His Arg Ile Cys                85   #                90   #                95Cys Arg Ala Cys Arg Ile Lys Lys His Lys Le #u Arg Ile Tyr Phe Glu            100       #           105       #           110Ser Lys Lys Phe Leu Leu Leu Leu Tyr Leu Va #l Leu His Phe Leu Phe        115           #       120           #       125Ser Ser Lys Ile Asn Thr Leu Leu Gln Asp Ph #e Ser Leu    130               #   135               #   140 <210> SEQ ID NO 124<211> LENGTH: 437 <212> TYPE: DNA <213> ORGANISM: Homo sapiens<400> SEQUENCE: 124gtcaccccag ccatgaggac ctttgccctc ctcactgcca tgcttctcct gg#tggccctg     60taggctcagg cagagccact tcaggcaaga gctgatgaag ctgcagccca gg#agcagcct    120ggagcagatg atcaggaaat ggctcatgcc tttacatggc atgaaagtgc cg#ctcttccg    180ctttcagtca gactcagcga gaggcttgag gtgcatttgc ggaagaggaa tt#tgccgttt    240gttataacgt cgctttgggt cctgcgcctt tcgtggtaca ctccaccgga tc#tgctgccg    300ctgagcttgc agaatcaaga aacataagct cagaatttac tttgagagtt aa#aagaaatt    360cttgttactc ctgtaccttg tcctccattt ccttttctca tccaaaataa at#accttgtt    420 gcaagatttc tctcttt              #                  #                   #  437 <210> SEQ ID NO 125 <211> LENGTH: 141<212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 125Val Thr Pro Ala Met Arg Thr Phe Ala Leu Le #u Thr Ala Met Leu Leu 1               5   #                10   #                15Leu Val Ala Leu Ala Gln Ala Glu Pro Leu Gl #n Ala Arg Ala Asp Glu            20       #            25       #            30Ala Ala Ala Gln Glu Gln Pro Gly Ala Asp As #p Gln Glu Met Ala His        35           #        40           #        45Ala Phe Thr Trp His Glu Ser Ala Ala Leu Pr #o Leu Ser Ser Asp Ser    50               #    55               #    60Ala Arg Gly Leu Arg Cys Ile Cys Gly Arg Gl #y Ile Cys Arg Leu Leu65                   #70                   #75                   #80Arg Arg Phe Gly Ser Cys Ala Phe Arg Gly Th #r Leu His Arg Ile Cys                85   #                90   #                95Cys Arg Ala Cys Arg Ile Lys Lys His Lys Le #u Arg Ile Tyr Phe Glu            100       #           105       #           110Ser Lys Lys Phe Leu Leu Leu Leu Tyr Leu Va #l Leu His Phe Leu Phe        115           #       120           #       125Ser Ser Lys Ile Asn Thr Leu Leu Gln Asp Ph #e Ser Leu    130               #   135               #   140

What is claimed is:
 1. A circular peptide comprising two linkednonapeptides, wherein one nonapeptide has the sequence set forth in SEQID NO:74 and one nonapeptide has a sequence selected from the groupconsisting of SEQ ID NO:19 to SEQ ID NO:64: and SEQ ID NO:74 to SEQ IDNO:119.
 2. The peptide according to claim 1 wherein one nonapeptide hasthe sequence set forth in SEQ ID NO:19 and one nonapeptide has thesequence set forth in SEQ ID NO:74.
 3. The peptide according to claim 1and a pharmaceutically acceptable excipient.
 4. A method for killingmicrobial organisms, the method comprising: administering an effectivedose of a peptide according to claim 1 to said microbial organisms. 5.The method according to claim 4, the method comprising: administeringsaid peptide as a therapeutic agent to a patient with an establishedmicrobial infection.
 6. A method for administering retrocyclin as aprophylactic agent, the method comprising: administering a peptideaccording to claim 1 to a patient to inhibit a microbial infection in apatient at risk of developing such infection.